ALBERT

Description: Multipotent mesenchymal stromal cells (MSCs) have immunomodulatory properties and have been successfully used for treatment of autoimmune diseases and acute or chronic graft-versus-host disease. Therapy with MSCs is not always effective. It has been shown that MSCs immunomodulatory properties can be improved by means of various agents, such as IFN-g, TNF-a, IL-17. After 4 hours of IFN-g exposure the expression level of immunomodulatory genes increased - IDO1 300, CSF1 - 7, and IL6 - 2.4 times. MSCs typically express low levels of MHC class I, and no MHC class II or co-stimulatory molecules (e.g., B7-1, B7-2, or CD40), making them partially immunoprivileged. However, treatment with IFN-g leads to increased expression of HLA-DR antigens on MSCs. After injection to the patient the characteristics of MSCs differ from those which have been studied in culture due to their interactions with other cells in the bloodstream and tissues. In this study the model of MSCs and MSCs treated with IFN-g (IFN-g-MSC) interactions with allogeneic lymphocytes in vitro was developed. The aim of the study was to identify the changes in MSCs and IFN-g-MSCs characteristics after co-cultivation with lymphocytes in vitro in dynamics. Materials and methods MSCs were isolated from 13 bone marrow (BM) samples used for allogeneic hematopoietic cells transplantation and cultured by a standard method in aMEM with 10% fetal bovine serum (FBS). MSCs on 2-3-d passages were seeded 105 cells per flask with 25 cm2 bottom area and a day later 500 units/mL of IFN-g were added for 4 hours to half of the cultures. Then the media was changed on RPMI-1640 with 10% FBS. Some cultures were seeded with 106 allogeneic lymphocytes, to half of these cultures 5 mg/ml phytohemagglutinin (PHA) was added for lymphocytes activation. All flasks were cultured up to 4 days at 37°C and 5% CO2. After 1, 2, 3 and 4 days lymphocytes were washed from MSCs. MSCs were removed from the flasks with trypsin and the number of viable cells was determined by dye exclusion method (trypan blue). For each of the MSCs cultures the mean fluorescent signal intensity level (MFI) of HLA-DR was determined by direct immunofluorescent staining with anti-HLA-DR APC (BD Pharmingen) antibodies and measured on flow cytometer BD FACS Canto II (BD Biosciences, USA). Data are presented as mean ± standard error. Statistical analysis was performed using Student's t-test (considered reliable p

Description: The stromal microenvironment regulating hematopoiesis in patients with hematologic malignancies undergoes significant alterations. The changes in the concentration of colony-forming fibroblast units (CFU-F) in the bone marrow (BM) and disruption in the functioning of multipotent mesenchymal stromal cells (MSCs) are shown in many studies for patients with acute leukemia. Often it is not possible to distinguish the cause of changes in stromal progenitor cells after treatment: interaction with tumor cells or the effects of therapy. Most of the patients with diffuse large B-cell lymphoma (DLBCL) do not have BM involvement. It was assumed that the properties of MSCs in these patients were not changed, and so this could be an attractive model for investigation the effect of antitumor drugs on human BM stromal microenvironment. The aim of the study was to compare the properties of MSCs in patients with DLBCL in the onset of the disease and a month after the end of therapy. Methods The study included 20 patients with DLBCL (11 male, 9 female) aged 42-60 years in the onset of the disease and a month after the end of treatment. 3-5 ml of BM were collected during diagnostic punctures after informed consent. MSCs and CFU-F were cultured by standard methods. The total MSCs production, the doubling-population level per day, the concentration of CFU-F, the relative gene expression level (REL) in MSC by real-time PCR and the mean fluorescence intensity (MFI) by flow cytometry were analyzed. The control group included 31 donors of the corresponding age. The analysis of MSCs secretome was carried out using the LC-MS/MS analysis TripleTOF 5600+ mass spectrometer with a NanoSpray III ion source coupled to a NanoLC Ultra 2D+ nano-HPLC System. Results The total cell production for 4 passages in primary patients' MSCs was significantly higher than in donors (11.4 ± 2 x 106 per flask versus 6.9 ± 1.1, p = 0.04). It remained elevated after therapy (10.2 ± 1.5). At the same time, the MSCs population-doubling level per day was significantly decreased in patients in comparison with donors (0.6 ± 0.03 vs. 0.4 ± 0.04, p

Description: Introduction In acute and chronic leukemia, the changes in the basic properties of multipotent mesenchymal stromal cells (MSCs), including morphology, immunomodulatory abilities, and the expression of various genes, were described. The aim of the investigation was to study the ability of MSCs derived from the bone marrow (BM) of patients with acute lymphoblastic (ALL), myeloid (AML) leukemia and chronic myeloid leukemia (CML) to maintain normal hematopoietic progenitor cells. Methods The study included 14 patients with ALL, 25 with AML and 15 with CML. All work was conducted in accordance with the Declaration of Helsinki (1964). BM was aspirated from the patient during diagnostic punctures before the treatment and for patients with acute leukemia 37 and 100 days; for patients with CML on average at the 125th day and 220th day after the start of the treatment. The BM samples of 22 healthy donors were used as controls. MSCs were derived from 5-10 ml of BM and cultivated at the density 3 х 106 cells in T25 culture flasks in aMEM with 10% FCS. The ability of MSCs from patients to maintain normal hematopoietic precursor cells - coble stone area forming cells (CAFC) was performed by the limiting dilution method. One day prior to assay, MSCs from donors and patients were explanted with 1000 cells per well of 96-well plates. As a control, the cell line MS5 supporting growth of hematopoietic precursor cells was used. On the following day, with complete medium change, BM cells from healthy donors were implanted in four serial dilutions. The frequency of CAFC in the normal BM was performed using Poisson's equation and presented as the percent of control. The total RNA was extracted from MSCs at passage 1 using the standard method. The relative gene expression levels (REL) were determined by normalizing the expression of each target gene to the levels of beta-actin and glyceraldehyde 3-phosphate dehydrogenase and calculated using the ΔΔCt method. Results Total cell production of MSCs from ALL (5.6±1.5) x 106 and AML (5.4±1.0) x 106 patients decreased at the moment of diagnosis, whereas the production of MSCs from CML (7.9±1.9) x 106 patients did not differ from the donors (7.1±1.04) x 106. Reduced cell production is likely associated with a significant decrease in the expression level of FGF2, VEGF, BGLAP and SOX9 genes (Table). The ability of MSCs derived from the BM of AML patients at the onset of the disease to maintain normal CAFC (59.3±6.8) was significantly decreased (p = 0.02) when compared to donors (74.9±9.5). At the end of the first course of chemotherapy, the ability to maintain CAFC in patients' MSCs reached that of the donors(80.7±5.8); 2 months later, the CAFC frequency on MSCs from AML patients doubled (107.9±18.4) in comparison with the start of the disease (p = 0.04). The ability of MSCs derived from ALL patients to support CAFC was lower than that of MSCs of AML patients both before treatment (57.8±12) and 37 days after the start (57.2±7.4). Three months after the initiation of treatment of these patients, the ability of MSCs to maintain CAFC recovered (73.7±13.3) and reached that of the donors' MSCs. MSCs of CML patients (100.8±9.9) at the disease onset maintained CAFC better than donors' MSCs. This ability increased with treatment at 125th day (109.6±14.4) and at 220th day (169.6) after the start of the treatment. The expression levels of LIF, IGF1, IL6, CSF1 increased significantly in CML-derived as well as AML-derived MSCs, but changes were more pronounced in the case of CML. Table. REL of different genes in MSCs derived from patients at the onset of the disease. Gene AML ALL CML Donors FGF2 2.8±0.2* 2.0 ±0.4* 4.2±0.5 6.0±0.9 VEGF 1.5±0.1* 1.5±0.2* 1.7±0.2 2.5±0.9 LIF 9.1±1.3* 7.1±2.3* 21.8±7.5 2.2±0.4 IL6 12.7±1.8* 11.3±4.4 22.5±4.8* 6.2±1.8 CSF1 3.6±0.9* 2.1±1.2 0.96±0.1* 0.7±0.1 IGF1 1.3±0.3* 1.49±0.7 2.6±0.5* 0.6±0.1 BGLAP 0.6±0.2* 0.8±0.4* 1.7±0.4 2.7±0.1 SOX9 1.2±0.1* 0.9±0.3* 1.2±0.2 1.7±0.2 (*significantly different from donors) Conclusions Functional changes in MSCs, which are the part of BM stromal microenvironment and, in particular, a niche for the HSCs was revealed. The ability of MSCs to support CAFC is dramatically changed in patients with hematological malignancies; the nature of the functional alterations of MSCs depends on the diagnosis. In cases of acute leukemia, MSCs' ability to maintain CAFC normalized with the treatment, whereas the strengthening of this capacity was revealed in cases of CML. Disclosures Turkina: Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis Pharma: Consultancy.

Description: Introduction Multipotent mesenchymal stromal cells (MSCs) differentiate into all mesenchymal lineages, regulate hematopoietic stem cells, and also take part in immunomodulation. MSCs are damaged in patients with leukemia. Most of the patients with DLBCL do not have bone marrow (BM) involvement. Despite the absence of proved BM damage in DLBCL patients, the properties of MSCs are changed. We aimed to analyze secretome and transcriptome of MSCs derived from BM of DLBCL patients without BM involvement. Methods The study included 16 DLBCL patients (7 males and 9 females), of which 6 were 42-60-year-old in the onset of the disease and a month after the end of treatment with NHL-BFM90; 10 were 48-78-year-old in complete remission for 6-14 years (5 received CHOP and 5 NHL-BFM90 treatment). Control group included 5 healthy donors (3 males, 2 females), median age 37. During diagnostic punctures BM was collected after informed consent. MSCs were cultured by standard method. Confluent MSCs layers after 1 passage were cultivated in serum-free RPMI1640 without phenol red for 24 hours; supernatants were studied for secretome and cells for transcriptome. The analysis of MSCs secretome was carried out using the LC-MS/MS analysis (TripleTOF 5600+ mass spectrometer with a NanoSpray III ion source coupled to a NanoLC Ultra 2D+ nano-HPLC System. Total RNA was isolated, applying standard procedures, from MSCs. Next-generation sequencing of complementary DNA libraries of polyA-enriched RNA was performed with Illumina HiSeq. Raw RNA-seq data were processed using STAR. Gene expression was compared using the limma R/Bioconductor package. Results The total cell production for 4 passages in primary patients' MSCs was higher than in donors (26.6 ± 2 versus 10.1 ± 4.4 x 106 per flask). It remained elevated regardless of the time passed after therapy. The patterns of secretome and transcriptome of patients' MSCs differed dramatically from the MSCs of healthy donors (Table). In MSCs of primary patients, the secretion and transcription of proteins involved in IL-17, TNF and Toll-like receptor signaling pathways, cytokine-cytokine receptor interaction, cytokine-mediated signaling pathway, cellular response to cytokine stimulus, regulation of signaling receptor activity, regulation of neutrophil chemotaxis, inflammatory and acute inflammatory response and its regulation, leukocyte activation involved in immune response, immune system process, extracellular matrix organization were elevated. Secretion and transcription of cytokines and chemokines (IL6, IL4, LIF, TNFa, CXCL1 and CXCL3), taking part in hematopoiesis regulation were increased in primary patients MSCs. One month after treatment, secretion of 332 proteins was decreased, only 2 of them (DKK1 and FKBP7) were previously overexpressed in primary patients. Many years after the end of both variants of treatment, the secretion and transcription of 32 proteins participating in the same pathways as before treatment remains elevated compared with healthy donors. In addition, the complement and coagulation cascades became upregulated. In MSCs of all patients, regardless of therapy and remission duration , expression/ secretion of following genes/proteins: ACAN, COL1A, MMP3, TGFb1, NDNF, CANX, LAP3, MGP, SERPINB2, STC1,TFPI,TMEM132A, BMP2, CFH, HILPDA, IDO1, IL1B, ITGA2, JUN, LMO2, MMP13, MMP3, TNFRSF1B,TNFSF4 was increased. Some of these proteins take part in bone and cartilage formation, hematopoietic stem cells regulation, blood coagulation and inflammation. These changes in secreted proteins reflect the response of MSCs at the organism level to the tumor presence. Moreover, NUCKS1 overexpression was observed in MSCs of all patients. This nuclear casein kinase plays a significant role in modulating chromatin structure and regulates replication, transcription, and chromatin condensation. Furthermore, this protein contributes to the susceptibility, occurrence, and development of several types of cancer and other diseases. NUCKS1 is considered to be a potent marker for such diseases. Conclusion The presence of a lymphoid tumor without BM involvement in the body leads to irreversible changes in the BM MSCs, thus affecting a lot of biological processes and signaling pathways, independent of the treatment and duration of complete remission. The work were supported by the Russian Foundation for Basic Research, Project No. 17-00-00170. Disclosures No relevant conflicts of interest to declare.

Description: Introduction. Stromal microenvironment of the bone marrow (BM) is essential for normal hematopoiesis; the very same cells are involved in the interaction with the leukemic stem cells. The aim of the study was to reveal the alterations in stromal microenvironment of patients in debut and after the therapy using multipotent mesenchymal stromal cells (MSC) as a model. Methods. MSC of patients with acute myeloid leukemia (AML, N=32), acute lymphoblastic leukemia (ALL, N=20), chronic myeloid leukemia (CML, N=19), and diffuse large B-cell lymphoma without BM involvement (DLBCL, N=17) were isolated by standard method from the patients' BM. Each BM sample was acquired during diagnostic aspiration after the informed signed consent was obtained from the patient. Groups of BM donors comparable by age and gender were used as controls for each nosology. Gene expression was analyzed with real-time RT-PCR. The significance of differences was evaluated with Mann-Whitney U-test. Results. The results of gene expression analysis are summarized in Table. The expression of genes regulating hematopoietic stem and precursor cells (JAG1, LIF, IL6) was significantly upregulated in MSC of the patients in debut, except for DLBCL. The latter was characterized with upregulation of osteogenic marker gene SPP1 and downregulation of FGFR1 gene. The upregulation of the expression of genes regulating proliferation of stromal cells (PDGFRA, FGFR1) and adipogenic marker gene (PPARG) was common for AML and CML. Both acute leukemias were characterized by the upregulation of genes associated with inflammation and regulation of hematopoietic precursors (CSF1, IL1B, IL1BR1) and by the downregulation of chondrogenic differentiation marker gene (SOX9). CML and DLBCL demonstrated the upregulation of FGFR2. BM of the DLBCL patients did not contain any malignant cells; nevertheless, stromal precursors from the BM were significantly affected. This indicates the distant effects of DLBCL malignant cells on the patients' BM. Myeloid malignancies seem to affect MSC more profoundly then lymphoid ones. Effect of leukemic cells on stromal microenvironment in case of myeloid leukemia was more pronounced. The treatment significantly affected gene expression in MSC of patients. In all studied nosologies the IL6 gene expression was upregulated, which may reflect the inflammation processes ongoing in the organism. The expression of LIF was upregulated and ICAM1, downregulated in MSCs of AML, ALL, and CML patients. In the MSC of patients with AML, who had received the highest doses of cytostatic drugs to achieve remission, a significant decrease in the expression of most studied genes was found. In patients with ALL with long-term continuing treatment in combination with lower doses of drugs, IL1B expression was increased, while the decrease in expression was detected for a number of genes regulating hematopoietic stem cells (SDF1, TGFB1), differentiation and proliferation (SOX9, FGFR1, FGFR2). Treatment of CML patients is based on tyrosine kinase inhibitors in doses designed for long-term use, and is less damaging for MSC. The upregulation of TGFB1, SOX9, PDGFRA genes and downregulation of IL1B gene was revealed. MCS of DLBCL patients, unlike the other samples, were analyzed after the end of treatment. Nevertheless, significant upregulation of IL8 and FGFR2 genes was found. Thus, both the malignant cells and chemotherapy affect stromal precursor cells. The changes are not transient; they are preserved for a few months at least. MSCs comprise only a minor subpopulation in the BM in vivo. When expanded in vitro, they demonstrate significant changes between groups of patients and healthy donors. Conclusions. Leukemia cells adapt the stromal microenvironment. With different leukemia, the same changes are observed in the expression of genes in MSC. MSC of patients with acute forms have a lot of changes which coincide among these two diseases. MSC of AML patients are most affected both in debut and after the therapy. Treatment depends on the nosology and in varying degrees changes the MSC. This work was supported by the Russian Foundation for Basic Research, project no. 17-00-00170. Disclosures Chelysheva: Novartis: Consultancy, Honoraria; Fusion Pharma: Consultancy. Shukhov:Novartis: Consultancy; Pfizer: Consultancy. Turkina:Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy; Novartis: Consultancy, Speakers Bureau; fusion pharma: Consultancy.