ALBERT

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.