Acute Myeloid Leukemia Causes Serious and Partially Irreversible Changes in Secretomes of Bone Marrow Multipotent Mesenchymal Stromal Cells

In patients with acute myeloid leukemia (AML), malignant cells modify the properties of multipotent mesenchymal stromal cells (MSCs), reducing their ability to maintain normal hematopoiesis. The aim of this work was to elucidate the role of MSCs in supporting leukemia cells and the restoration of normal hematopoiesis by analyzing ex vivo MSC secretomes at the onset of AML and in remission. The study included MSCs obtained from the bone marrow of 13 AML patients and 21 healthy donors. The analysis of proteins contained in the MSCs-conditioned medium demonstrated that secretomes of patient MSCs differed little between the onset of AML and remission; pronounced differences were observed between MSC secretomes of AML patients and healthy donors. The onset of AML was accompanied by a decrease in the secretion of proteins related to ossification, transport, and immune response. In remission, but not at the onset, secretion of proteins responsible for cell adhesion, immune response, and complement was reduced compared to donors. We conclude that AML causes crucial and, to a large extent, irreversible changes in the secretome of bone marrow MSCs ex vivo. In remission, functions of MSCs remain impaired despite the absence of tumor cells and the formation of benign hematopoietic cells.


Introduction
The bone marrow microenvironment plays a central role in maintaining the dynamic balance between hematopoietic stem and progenitor cells (HSPCs) self-renewal, differentiation, quiescence, and proliferation in homeostatic and pathologic conditions [1]. This microenvironment is composed of multiple cell types, the most prominent of which are considered to be specific subsets of multipotent mesenchymal stromal cells (MSCs) or derived from them [2]. Bona fide MSCs are rare elements in the bone marrow (0.01%) [3], which are able to differentiate into all types of stromal cells (fibroblasts, osteoblasts, adipocytes, etc.). Within the bone marrow, MSCs are prominently involved in orchestrating the behavior of hematopoietic stem and progenitor cells, ensuring a lifelong blood supply [2,4]. The MSCsrelated cells, in particular the CAR cells (CXCL12-abundant reticular cells), the nestin+ cells, and the CD146+ cells, play a major role in the communication between the bone the MSC secretome in remission, although it differs from the one at the onset of the disease, does not show significant recovery and still differs substantially from the MSC secretome in healthy donors.

Results
MSCs were obtained from bone marrow mononuclear cells collected from patients at diagnosis (referred to as AML-MSCs), at the time of remission (R-MSCs), and from agematched healthy donors (D-MSCs). A summary of the data is presented in Figure 1. Differential expression was calculated on normalized log2 ratios. Differences were considered significant at p < 0.05, (FC ≥ 2).

Comparison of AML-MSC and D-MSC Secretomes
The work analyzed 2833 proteins identified in the secretomes of the studied MSCs. Among these, 685 proteins significantly differed in AML-MSCs, R-MSCs, and D-MSCs. Secretion levels for 533 protein were different in AML compared to donors but not in remission (Table S1, Supplementary Materials).

Comparison of AML-MSC and D-MSC Secretomes
The work analyzed 2833 proteins identified in the secretomes of the studied MSCs. Among these, 685 proteins significantly differed in AML-MSCs, R-MSCs, and D-MSCs. Secretion levels for 533 protein were different in AML compared to donors but not in remission (Table S1, Supplementary Materials). AML-MSCs lost secretion of five proteins compared to D-MSCs: these are proteins associated with the extracellular region (NCAM1, F10, CST6, A1BG, LOXL4).
When comparing upregulated genes in AML-MSCs versus D-MSCs-AML-MSCs secreted 141 proteins significantly more actively than D-MSCs. Among them are proteins that are important for maintaining hematopoiesis and the immune response, forming relationships-HLA-A, MIF, FN1, COL8A1, POSTN, IGFBP5, CD44, LGALS3 и ANXA1, CXCL12 (Tables S2 and S3  GO-term enrichment analysis for compartment, biological process, and cellular component of AML-MSCs versus D-MSCs is presented in Table 1. Many different proteins involved in various spheres of cell activity are changed in patients' MSCs. Proteins involved simultaneously in various processes change their expression in AML-MSCs (Figure 2B,C). The changes are associated with the extracellular space, matrix, exosomes, and vesicles. Many changes are associated with the involvement of MSCs in the maintenance of hematopoietic cells and the immune response. Antigen processing and presentation of exogenous peptide antigen, Antigen processing and presentation of exogenous peptide antigen via MHC class I, tap-dependent, Cytokine-mediated signaling pathway, Regulated exocytosis, Proteasomal ubiquitinindependent protein catabolic process, Cellular response to cytokine stimulus, Vesicle- GO-term enrichment analysis for compartment, biological process, and cellular component of AML-MSCs versus D-MSCs is presented in Table 1. Many different proteins involved in various spheres of cell activity are changed in patients' MSCs. Proteins involved simultaneously in various processes change their expression in AML-MSCs ( Figure 2B,C). The changes are associated with the extracellular space, matrix, exosomes, and vesicles. Many changes are associated with the involvement of MSCs in the maintenance of hematopoietic cells and the immune response. Antigen processing and presentation of exogenous peptide antigen, Antigen processing and presentation of exogenous peptide antigen via MHC class I, tap-dependent, Cytokine-mediated signaling pathway, Regulated exocytosis, Proteasomal ubiquitin-independent protein catabolic process, Cellular response to cytokine stimulus, Vesicle-mediated transport, Immune system process, Secretion, interleukin-1-mediated signaling pathway, Cellular process

Comparison of R-MSCs and D-MSCs Secretome
Comparison of the secretomes of R-MSCs and D-MSCs revealed significant differences in 550 proteins. Obviously, there was no recovery of secreted proteins when remission was achieved. There was no recovery of secretion of proteins whose expression was lost in AML-MSCs (NCAM1,  GO-term enrichment analysis for the biological process of R-MSCs versus D-MSCs is presented in Table 2. Thus, upon reaching remission, the composition of the R-MSCs secretome does not recover, and it continues to differ from the secretome of D-MSCs.

Comparison of AML-MSCs and R-MSCs Secretome
Comparison of the secretomes of AML-MSCs and R-MSCs revealed similar and different proteins in secretion ( Figure 4 and Figure S3, Supplementary Materials).
GO-term enrichment analysis for biological process of AML-MSCs versus R-MSCs presented in Table 3.    Secreted in AML-MSCs, not secreted in R-MSCs: 255 proteins, significant only 3 proteins-UCL1-Mucin-like protein 1; May play a role as a marker for the diagnosis of metastatic breast cancer; SSBP1-Single-stranded DNA-binding protein, mitochondrial; this protein binds preferentially and cooperatively to ss-DNA. Probably involved in mitochondrial D; UBE2I-SUMO-conjugating enzyme UBC9; Accepts the ubiquitin-like proteins SUMO1, SUMO2, SUMO3, and SUMO4 from the UBLE1A-UBLE1B E1 complex.
Secreted in R-MSCs, not secreted in AML-MSCs: 405 proteins, significant only 3: F10-Coagulation factor X; Factor Xa is a vitamin K-dependent glycoprotein that converts prothrombin to thrombin in the presence of factor Va; CTGF-Cellular communication network factor 2; Connective tissue growth factor; Major connective tissue mitoattractant secreted by vascular endothelial cells; LAMA2-Laminin subunit alpha-2; Binding to cells via a high-affinity receptor, laminin is thought to mediate the attachment, migration, and organization of cells.
The number of proteins with different secretion in AML-MSCs and R-MSCs compared to D-MSCs and their main functions is presented in Figure 1, Table 4, Tables S2 and S3 Supplementary Materials. Table 4. Secretion differences between AML-MSCs and R-MSCs versus D-MSCs. GO-Gene Ontology.

Elements Pathway Database Term Description
Equally expressed compared to D-MSCs Higher in R-MSCs and AML-MSCs versus D-MSCs GO Biological process Extracellular matrix assembly, Platelet degranulation, Chondrocyte differentiation, interleukin-12-mediated signaling pathway, Platelet-derived growth factor receptor signaling pathway, Osteoclast differentiation, Regulation of epithelial to mesenchymal transition, Bone development, Cellular response to transforming growth factor beta stimulus, Cell adhesion, Angiogenesis, Blood coagulation, Mesenchymal cell differentiation, Secretion, Response to growth factor, Negative regulation of canonical WNT signaling pathway, Cell activation involved in immune response, Aging, Vesicle-mediated transport, Posttranscriptional regulation of gene expression, Regulation of translation, Immune system process, Regulation of cell death, Cellular protein modification process, Signaling

Lower in R-MSCs and AML-MSCs versus D-MSCs
Biological process Response to interleukin-1, Extracellular matrix organization, Leukocyte migration, Response to hypoxia, Angiogenesis, Cytokine-mediated signaling pathway, Blood vessel morphogenesis, Cellular response to cytokine stimulus, Leukocyte mediated immunity, Cell adhesion, Secretion, Vesicle-mediated transport, Immune system process, Cell differentiation  GO-term enrichment analysis for the biological process of AML-MSCs and R-MSCs versus D-MSCs is presented in Table 4.

Discussion
The tumor microenvironment is considered nowadays as one of the main players in cancer development and progression. AML microenvironment is highly complex and includes MSCs, their progeny, and a large list of extracellular matrix proteins and soluble factors secreted by AML-MSCs.
Comparative studies of the MSC secretome revealed large differences between the secretomes of AML-MSCs and D-MSCs, as well as the absence of the full restoration of the MSC secretome profile after treatment and upon reaching remission. D-MSCs did not secrete PPARGC1A -Peroxisome proliferator-activated receptor gamma coactivator 1-alpha. This protein is involved in the immune response and is a pivotal transcriptional coactivator regulating mitochondrial biogenesis and metabolism [30]. PPARGC1A greatly increases the transcriptional activity of PPARG and thyroid hormone receptors.
As compared to D-MSCs, the secretion of six proteins was not revealed in AML-MSCs. One of them is NCAM1-Neural cell adhesion molecule 1 (CD56), representing a transmembrane glycoprotein modulating cell-cell and cell-matrix interactions. NCAM1 expression is strongly associated with constitutive activation of the MAPK-signaling pathway, regulation of apoptosis, or glycolysis [31]. Bone marrow-derived MSCs from NCAM-deficient mice exhibit the defective migratory ability and significantly impaired adipogenic and osteogenic differentiation potential. The mechanism governing NCAM1-mediated migration of MSCs involves the cross-talk between NCAM1 and fibroblast growth factor receptor (FGFR), which in turn activates MAPK/ERK signaling and, thereby, the migration of MSCs [32]. Clinical-grade human bone marrow MSCs were previously shown to express NCAM1 isoforms on their surface [33]. The presence of NCAM1 in a secreted fraction is likely to reflect either its inclusion in microvesicles produced by MSCs [34] or shedding by extracellular proteases [35]. Thus, the complete absence of secretion of this protein in AML-MSCs may indicate strongly reduced production of this protein and, thus major changes in cell-matrix interactions in these cells.
Another protein with strongly reduced secretion is CST6-Cystatin-E/M belongs to the type 2 cystatin family, which are mainly extracellular polypeptide inhibitors of cysteine proteases acting to prevent excessive proteolysis. Three proteases, including cathepsin B (CTSB), cathepsin L (CTSL), and legumain (LGMN), are known to be inhibited by CST6 in human cells. Several peptides mimicking the function of CST6 are able to suppress cancer cell-induced osteoclastogenesis and bone metastasis [36]. These findings reveal the CST6-CTSB signaling axis in osteoclast differentiation and provide a promising approach to treating bone diseases with CST6-based peptides [36]. Both recombinant CST6 protein and serum from patients with high CST6 significantly inhibited the activity of the osteoclast-specific protease cathepsin K and blocked osteoclast differentiation and function. Recombinant CST6 inhibited bone destruction in ex vivo and in vivo myeloma models [37]. Thus, the secretome of AML-MSCs, in contrast to normal MSCs, is unlikely to inhibit osteoclast differentiation.
A third protein is LOXL4 (Lysyl oxidase homolog 4), which may modulate the formation of a collagenous extracellular matrix. LOXL4 is an amine oxidase that is primarily involved in extracellular matrix remodeling. In vitro exposure of macrophages to LOXL4 invoked an immunosuppressive phenotype and activated programmed death ligand 1 (PD-L1) expression, which further suppressed the function of CD8 + T cells [38]. LOXL4 knockdown enhances tumor growth and lung metastasis through collagen-dependent extracellular matrix changes in triple-negative breast cancer [39]. Therefore, the not revealed secretion of LOXL4 in AML-MSCs could indirectly support the AML blast cells. The role of the remaining proteins not revealed in AML-MSC secretome-F10 and A1BG-is not obvious. It should be noted that all proteins mentioned above fail to restore their secretion level when remission is achieved. In particular, no secretion in R-MSCs is observed for NCAM1, secretion of CST6 is partially restored in 2 patients of 13, secretion of A1BG and LOXL4 restored in 1 patient of 13, while F10 secretion is restored in 5 patients of 13. The level of secretion of all proteins that appeared in remission remains very low. So AML-MSCs do not secrete several proteins important for maintaining normal functions.
GAS6 (Growth arrest-specific protein 6)-ligand for tyrosine-protein kinase receptors AXL, TYRO3, and MER, whose signaling is implicated in cell growth and survival, cell adhesion, and cell migration. GAS6 has important effects on hemostasis and inflammation [40]. Its deficiency affects various processes such as preventing apoptosis of endothelial cells during acidification, cytokine signaling, hepatic regeneration, gonadotropin-releasing hormone neuron survival and migration, platelet activation, or regulation of thrombotic responses. Decreased secretion of GAS6 and AXL by AML-MSCs was observed in some studies and resulted in a reduced ability of MSCs to proliferate [1]. COL6A1 (Collagen VI) is a major player in extracellular matrix biology since its deficiency alters extracellular matrix structure and biomechanical properties and leads to increased apoptosis and oxidative stress, decreased autophagy, and impaired muscle regeneration [41].
TGFB (Transforming growth factor beta) is a pleiotropic factor involved in many processes in the body associated with hematopoietic stem cells and the development of various diseases. The autocrine and paracrine effects of TGF-beta on tumor cells and the tumor microenvironment exert both positive and negative influences on cancer development [42].
PDGFA (Platelet-derived growth factor alfa) The classical PDGF polypeptide chains, PDGF-A and PDGF-B, are well-studied and known to regulate several physiological and pathophysiological processes, primarily acting on cells of mesenchymal or neuroectodermal origin [43]. It is secreted by melanoma cells and maintains the growth of malignant cells. Its deficiency may have a dual effect on MSCs and AML blasts [43].
PDGFRB (Platelet-derived growth factor receptor, beta) PDGFR-beta signaling is important for blood vessel formation and early hematopoiesis and has been implicated in a range of pathologies. Paracrine PDGF signaling triggers stromal recruitment in epithelial cancers and may be involved in epithelial-mesenchymal transition, affecting tumor growth, angiogenesis, invasion, and metastasis [44], while autocrine activation of PDGF signaling pathways is involved in gliomas, sarcomas, and leukemias. VCAM1 (Vascular cell adhesion molecule 1) was originally identified as a cell adhesion molecule that helps regulate inflammation-associated vascular adhesion and the transendothelial migration of leukocytes, such as macrophages and T cells. Recent evidence suggests that VCAM-1 is closely associated with the progression of various immunological disorders, including rheumatoid arthritis, asthma, transplant rejection, and cancer [45]. CFH (Complement factor H) is a central regulator of early alternative pathway activation by acting as a cofactor for factor I in the cleavage of C3b into iC3b [46]. Complement deficiencies within the mannose-binding lectin pathway generally lead to increased bacterial infections, and deficiencies within the alternative pathway usually lead to an increased frequency of Neisseria infections. However, factor H deficiency can lead to membranoproliferative glomerulonephritis and hemolytic uremic syndrome [47].
A comparison of secretomes of AML-MSC and D-MSCs identifies 137 proteins secreted at significantly higher levels by AML patients' MSCs. Proteins secreted at higher levels by AML-MSCs may indicate the possible activation of these cells. Interestingly, MSCs grown in culture for at least two passages exhibit the properties of activated cells, with this activation probably already occurring in the patient's bone marrow. Among the upregulated proteins are those that are important for maintaining hematopoiesis and the immune response, forming relationships-HLA-A, MIF, COL8A1, POSTN, IGFB5, CD44, LGALS3 и ANXA1, CXCL12.
Three proteins, namely HLA-A (HLA class I histocompatibility antigen) and B2M (beta-2-microglobulin), are involved in the presentation of foreign antigens. It is known that in patients with non-Hodgkin's lymphomas, plasma levels of b2 microglobulin and secreted HLA-ABC are increased [48]. Elevated levels of secreted HLA-I are found in the blood serum of patients with rheumatoid arthritis, systemic lupus erythematosus, and some viral infections [49]. It has been shown that with some solid tumors, the level of HLA-I in the blood serum of patients also increases [50]. There is evidence indicating that chemotherapy and targeted therapies are effective at enhancing HLA class I component expression and function in cancer cells [51]. It is assumed that increased secretion of HLA-I is associated with increased production of cytokines and activation of AML-MSCs in comparison with D-MSCs.
However, another protein-MIF (macrophage migration inhibitory factor)-is a proinflammatory cytokine involved in the innate immune response to bacterial pathogens. The expression of MIF at sites of inflammation suggests its role as a mediator in regulating the function of macrophages in host defense. MIF upregulation forms a pro-tumor microenvironment in response to hypoxia-induced factors and promotes pro-inflammatory cytokine production [52]. MIF activates CD44, which is elevated in AML-MSCs secretome. CD44 is an adhesion molecule that mediates the activation of the Src proto-oncogene protein family. MIF-activated CD44 is expressed in cells with dynamic proliferation, such as tumor cells [53]. The increased secretion of these two proteins indicates the maintenance of malignant cells by AML-MSCs. POSTN (periostin) may regulate multiple biological behaviors of tumor cells [54]. Periostin is a member of matricellular proteins that regulate a variety of biological processes in normal and pathological situations. Many members of this family, such as periostin, osteopontin (SPP1), or the CNN (Cyr61, CCN2, CCN3) family of proteins, have been shown to regulate key aspects of tumor biology, including proliferation, invasion, matrix remodeling, and dissemination to pre-metastatic niches in distant organs [55].
IGFBP5 (insulin-like growth factor binding protein 5) is a secreted protein involved in insulin-like growth factor signaling, a critical pathway in growth and development. IGFBP5 modulates matrix signaling and can increase fibrosis and adipogenesis [56]. Elevation of IGFBP5 secretion plays a role in cancer progression and could be associated with poor prognosis.
LGALS3 (Galectin 3) is a member of the galectin family. It is predominantly located in the cytoplasm; however, it shuttles into the nucleus and is also a secreted protein. It serves important functions in numerous biological processes, including cell growth, apoptosis, pre-mRNA splicing, differentiation, transformation, angiogenesis, inflammation, fibrosis, and host defense [57]. Galectin-1, galectin-3, and galectin-9 secreted by MSCs prevent proliferation and induce apoptosis of activated Th1-and Th17-lymphocytes and CD8 + T-lymphocytes, promote the survival of naive T-lymphocytes, generation of tolerogenic dendritic cells and activation of Tregs [58]. Increased secretion of galectins also indicates changes in MSCs associated with the presence of blast cells in the bone marrow in AML patients.
CXCL12-stromal-derived factor 1 (SDF1) is a chemokine belonging to a family of small cytokines with chemotactic activity and is responsible for homing of hematopoietic cells and responsive to pro-inflammatory stimuli. Elevated serum levels of CXCL12 were found to be associated with increased osteoclast formation and bone loss in myeloma patients, and that targeted disruption of the CXCL12/CXCR4 axis inhibited osteolysis in a murine model of myeloma-associated bone loss [59]. In addition to bone marrow stromal cells, circulating plasma cells in multiple myeloma also produce CXCL12 and the high levels of CXCL12 in this disease are involved in various pathological processes [60]. Enhanced secretion of CXCL12 by AML-MSCs and R-MSCs (in the latter case at lower levels and not statistically significant) versus D-MSCs indicates a substantial but not complete recovery of the secretion of this chemokine. The interaction of CXCL12 and its receptors subsequently induces downstream signaling pathways with broad effects on chemotaxis, cell proliferation, migration, and gene expression. Accumulating evidence suggests that the CXCL12/CXCR4/CXCR7 axis plays a pivotal role in tumor development, survival, angiogenesis, metastasis, and tumor microenvironment [61].
In the remission of the disease, there is little recovery of MSCs secretome. The 220 proteins are secreted at lower levels compared to D-MSCs in both AML-MSCs and R-MSCs, while the secretion of 104 proteins is uniquely reduced in AML-MSCs and 88 proteins-in R-MSCs. The 86 proteins that were upregulated in AML-MSCs compared to D-MSCs remain elevated in remission, with R-MSCs over-secreting 37 more unique proteins compared to D-MSCs. These data indicate strong changes in the secretome associated not only with the primary adaptation of the stromal microenvironment to tumor cells but also with the damaging effect of chemotherapy. The differences in protein secretion between AML-MSCs and R-MSCs are significantly smaller as compared to their differences to D-MSCs.
It should also be noted that in a number of instances, the secretion of the most important proteins associated with the processes of protein degradation (ubiquitin proteasome system), autophagy, ferroptosis, the translation process, the organization of the extracellular matrix, and others does not change unidirectionally, and many proteins involved in these processes change the level of secretion.
The above findings indicate that changes in MSC secretome induced by AML are largely irreversible. Of note, the aging of normal MSCs ex vivo is also considered to be irreversible and results in senescent phenotype. According to some studies, AML-MSCs, both in culture and in vivo, are reminiscent of normal senescent MSCs [62,63]. We may hypothesize that extensive proliferation inherent to AML cells induces rapid wear-out and exhaustion of AML niche MSCs that support a very intense leukemia cell metabolism. If so, this is likely to result in accelerated aging of AML-MSCs in bone marrow, a process that should be additionally enhanced by chemotherapy treatments.
A significant number of ribosomal proteins and translation initiation factors are reduced or absent in the secretome of AML-MSCs compared to donor MSCs. Whether this finding signifies the reduced translation efficiency in AML-MSCs remains to be clarified. It might be highly indicative, though, that a recent study demonstrated the loss of regulation of protein synthesis and reduction of ribosomal protein levels in senescent MSCs [64].
Changes in the secretome of MSCs in AML patients may be related to the fact that leukemic stem cells survive in the bone marrow of patients after chemotherapy and bone marrow transplantation and may begin to proliferate and cause a relapse of the disease. It has been suggested that MSCs have the ability to form a cancer stem cell niche in which tumor cells can preserve the potential to proliferate and sustain the malignant process [65]. Our data, although not directly pinpointing specific changes in the AML MSC secretome to the disease recurrence, open up opportunities for further studies of the leukemic bone marrow microenvironment that may be associated with relapse.
Finally, it would be important to reiterate that, according to numerous publications, AML-educated bone marrow niche subverts normal hematopoiesis and cooperates with leukemia cells. Combined with the major finding of this study demonstrates that changes in MSC secretome induced by AML are largely irreversible; this may indicate that even in remission, MSCs retain their "subverted" phenotype and remain the "enemy within" the patient's organism. This may, in turn, open up new therapeutic strategies aimed specifically at this "enemy within". It should be noted that traditional therapies targeting leukemic cells have failed to improve long-term survival rates, and the bone marrow niche is thus becoming a promising source of potential therapeutic targets, particularly for relapsed and refractory AML [66]. The data of this study on differences between D-MSC, AML-MSC, and R-MSC secretomes may provide important clues to developing new therapeutic strategies. These therapies may include pro-apoptotic agents, microenvironment targeting molecules, cell cycle checkpoint inhibitors, and epigenetic regulators [67]. In addition, one could envisage approaches directed towards reverse in vivo education of subverted MSCs using exosomes and microvesicles derived from normal MSCs. An alternative strategy may involve attempts to replace AML-subverted MSCs in the niche with their normal counterparts.

Patients
The study included MSCs obtained from the bone marrow of 13 AML patients: (3 male, 10 female, median age 38) and 21 donors (10 male, 11 female, median age 35) that were used as control (Table 5 and Table S4 Supplementary Materials). All donors and patients signed informed consent.

MSCs
Donor MSCs for each patient were prepared from the bone marrow of a hematopoietic stem cell donor collected at the time of collection, as described [68]. For research purposes, MSCs from the same donors, after signing their informed consent, were further expanded as described [69].
The MSCs were derived from 2 to 8 mL of donor bone marrow. For the separation of mononuclear cells, the bone marrow was mixed with an equal volume of alpha-MEM (ICN) containing 0.2% methylcellulose (1500 cP, Sigma-Aldrich, St. Louis, MO, USA). After 40 min, most of the erythrocytes and granulocytes precipitated, while the mononuclear cells remained in the supernatant. The supernatant fraction was aspirated and centrifuged for 10 min at 450 g.
The cells from the sediment were resuspended in a standard culture medium that was composed of alpha-MEM supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 2 mM L-glutamine (ICN, Costa Mesa, CA, USA), 100 U/mL penicillin (Synthesis, Russia), and 50 µg/mL streptomycin (BioPharmGarant, Vladimir, Russia). The cells (3 × 10 6 ) were cultured in T25 culture flasks (Corning-Costar, Corning, NY, USA). After reaching confluency, the cells were washed with 0.02% EDTA (ICN, USA) in a physiological solution (Sigma-Aldrich, USA) and then detached by 0.25% trypsin (ICN, USA) treatment (Passage 0). For expansion, the cells were seeded at 4 × 10 3 cells per cm 2 of the flask growth area. The cultures were maintained under hypoxia conditions at 37 • C in 5% O 2 and 5% CO 2 . The number of harvested cells was counted directly; cell viability was checked by trypan blue dye exclusion staining.

Preparation of MSC-Conditioned Medium
MSCs at passages 2-3 were seeded at 4 × 10 3 cells per cm 2 into T175 flasks (Costar, USA). After attaining confluence (3-4 days), the flasks were washed five times with phosphate buffer without Ca 2+ /Mg 2+ (Invitrogen, Waltham, MA, USA) and then cultured for 24 h in RPMI 1640 medium without serum and phenol red (HyClone, USA). The conditioned medium was spun off at 400 g and frozen at −70 • C.

Proteomic Analysis of Secretomes
For proteomic analysis, secretome samples were thawed and passed through a 25 mm Syringe Filter 0.20 µm (GVS, Findlay, OH, USA), followed by the addition of Protease Inhibitor Mix and Acetonitrile (ACN) to a final concentration of 10%. The samples were then placed in an Amicon Ultra-15 centrifuge concentrator (Millipore, Burlington, MA, USA) with a nominal molecular weight cutoff of 3 kDa and centrifuged at 5000× g at 4 • C for 30 min. A total of 10 mL of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 3 mM MgCl 2 and 10% ACN was added to the concentrated samples, mixed, and centrifuged at 5000× g at 4 • C for 30 min. The eluates were discarded, and this step was repeated twice. A total of 1.5 mL of the concentrated sample was removed from the filter and transferred to a new tube, and TCEP (tris(2-carboxyethyl)phosphine) and CAA (Chloroacetamide) were added to the final concentrations of 5 and 30 mM, respectively. Cysteine reduction and alkylation were achieved by incubation of the sample at 80 • C for 10 min. Proteins were precipitated by the addition of 9x volume of cold acetone and incubation at −20 • C overnight. The protein pellet was washed twice with cold acetone, followed by resuspension in 50 µL of the 100 mM Tris-HCl buffer (pH 8.5). Protein concentration was determined by BCA Assay Kit (Sigma-Aldrich, St. Louis, MO, USA). Trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega, Madison, WI, USA) was added to protein samples at a ratio of 1/100 w/w and incubated for 2 h at 37 • C. Then the second trypsin portion 1/100 w/w was added, and the sample was incubated at 37 • C overnight. Proteolysis was stopped by adding TFA to 1%. Peptides were dried in a SpeedVac (Labconco, Kansas City, MO, USA) and resuspended in 20 µL of 3% ACN, 0.1% TFA in MilliQ water. The peptide concentration was determined by BCA Assay Kit (Sigma-Aldrich, St. Louis, MO, USA).

LC-MS/MS Analysis
Proteomic analysis was performed on an Orbitrap Q Exactive HF-X (Thermo Fisher Scientific, Waltham, MA, USA) mass spectrometer equipped with a nano-electrospray (nano-ESI) source and a high-pressure nanoflow chromatograph UPLC Ultimate 3000 (Thermo Fisher) equipped with a lab-packed reverse-phase (Kinetex C18, 2.4 µm) column (100 µm × 500 mm). The temperature of the column was thermostatically controlled at 60 • C. Samples were loaded in buffer A (0.1% Formic acid) and eluted with a linear (180 min) gradient of 3 to 55% buffer B (0.1% Formic acid, 80% Acetonitrile) at a flow rate of 220 nL/min. Mass spectrometric data were stored during automatic switching between MS1 scans and up to 16 MS/MS scans (topN method). The target value for MS1 scanning was set to 3 × 10 6 in the range 390-1400 m/z with a maximum ion injection time of 45 ms and a resolution of 60,000. The precursor ions were isolated at a window width of 1.4 m/z. Precursor ions were fragmented by high-energy dissociation in a C-trap with a normalized collision energy of 30 eV. MS/MS scans were saved with a resolution of 15,000 at 400 m/z and a value of 2 × 10 5 for target ions with a maximum ion injection time of 50 ms.

Protein Identification and Bioinformatics Analysis
Raw LC-MS/MS data from Q Exactive HF mass-spectrometer were converted to .mgf peak lists with MSConvert (ProteoWizard Software Foundation, Palo Alto, CA, USA). For this procedure, we used the following parameters: "-mgf -filter peakPicking true [1,2]".
For thorough protein identification, the generated peak lists were searched with MASCOT (version 2.5.1, Matrix Science Ltd., London, UK) and X! Tandem (ALANINE, 2017.02.01, 2017.02.01, The Global Proteome Machine Organization) search engines against UniProt human protein knowledgebase with the concatenated reverse decoy dataset. The precursor and fragment mass tolerance were set at 20 ppm and 0.04 Da, respectively. Databasesearching parameters included the following: tryptic digestion with one [70] possible missed cleavage, static modification for carbamidomethyl (C), and dynamic/flexible modifications for oxidation (M). For X! Tandem, we also selected parameters that allowed a quick check for protein N-terminal residue acetylation, peptide N-terminal glutamine ammonia loss, or peptide N-terminal glutamic acid water loss. Result files were submitted to Scaffold 5 software (version 5.1.0) for validation and meta-analysis. We used the local false discovery rate scoring algorithm with standard experiment-wide protein grouping. For the evaluation of peptide and protein hits, a false discovery rate of 5% was selected for both. False positive identifications were based on reverse database analysis. We also set protein annotation preferences in Scaffold to highlight Swiss-Prot accessions among others in protein groups.
The interactions between identified differentially secreted proteins were analyzed using the STRING-db online service.

Statistics
Statistical analysis was performed using GraphPad Prism version 8-1 (GraphPad Software Inc., San Diego, CA, USA). Due to the non-normal distribution of the data, the Mann-Whitney U test was used for comparison. Differences were considered significant at p < 0.05.
PCA analysis was performed on scaled and centered protein counts within the R environment.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available from the corresponding author upon reasonable request.