Jelena Krstic

Transforming Growth Factor-Beta and Oxidative Stress Interplay: Implications in Tumorigenesis and Cancer Progression.

Transforming growth factor-beta (TGF-β) and oxidative stress/Reactive Oxygen Species (ROS) both have pivotal roles in health and disease. In this review we are analyzing the interplay between TGF-β and ROS in tumorigenesis and cancer progression. They have contradictory roles in cancer progression since both can have antitumor effects, through the induction of cell death, senescence and cell cycle arrest, and protumor effects by contributing to cancer cell spreading, proliferation, survival, and metastasis. TGF-β can control ROS production directly or by downregulating antioxidative systems. Meanwhile, ROS can influence TGF-β signaling and increase its expression as well as its activation from the latent complex. This way, both are building a strong interplay which can be taken as an advantage by cancer cells in order to increment their malignancy. In addition, both TGF-β and ROS are able to induce cell senescence, which in one way protects damaged cells from neoplastic transformation but also may collaborate in cancer progression. The mutual collaboration of TGF-β and ROS in tumorigenesis is highly complex, and, due to their differential roles in tumor progression, careful consideration should be taken when thinking of combinatorial targeting in cancer therapies.

Mesenchymal stem cells of different origin: Comparative evaluation of proliferative capacity, telomere length and pluripotency marker expression.

AIMS In vitro expansion changes replication and differentiation capacity of mesenchymal stem cells (MSCs), increasing challenges and risks, while limiting the sufficient number of MSCs required for cytotherapy. Here, we characterized and compared proliferation, differentiation, telomere length and pluripotency marker expression in MSCs of various origins. MAIN METHODS Immunophenotyping, proliferation and differentiation assays were performed. Pluripotency marker (Nanog, Oct-4, SOX-2, SSEA-4) expression was determined by immunofluorescence. Quantitative PCR was performed for relative telomere length (RTL) analyses, while expression of relevant genes for pluripotency markers, differentiation state (Cbfa1, human placental alkaline phosphatase, peroxisome proliferator activated receptor, Sox9 and Collagen II a1), and telomerase reverse transcriptase (hTERT) was determined by semiquantitative RT-PCR. KEY FINDINGS Peripheral blood MSCs (PB-MSCs) and umbilical cord MSCs (UC-MSCs) showed the highest, while periodontal ligament MSCs (PDL-MSCs) and adipose tissue MSCs (AT-MSCs) the lowest values of both the replication potential and RTL. Although MSCs from exfoliated deciduous teeth (SHEDs), PDL-MSCs and AT-MSCs showed higher mRNA expression of pluripotency markers, all MSCs expressed pluripotency marker proteins. SHEDs and PDL-MSCs showed prominent capacity for osteogenesis, PB-MSCs and UC-MSCs showed strengthened adipogenic differentiation potential, while AT-MSCs displayed similar differentiation into both lines. SIGNIFICANCE The MSCs populations derived from different sources, although displaying similar phenotype, exhibited high degree of variability regarding biological properties related to their self-renewal and differentiation capacity. These data indicate that for more accurate use in cell therapy, individualities of MSCs isolated from different tissues should be identified and taken into consideration when planning their use in clinical protocols.

Urokinase type plasminogen activator mediates Interleukin-17-induced peripheral blood mesenchymal stem cell motility and transendothelial migration

Mesenchymal stem cells (MSCs) have the potential to migrate toward damaged tissues increasing tissue regeneration. Interleukin-17 (IL-17) is a proinflammatory cytokine with pleiotropic effects associated with many inflammatory diseases. Although IL-17 can modulate MSC functions, its capacity to regulate MSC migration is not well elucidated so far. Here, we studied the role of IL-17 on peripheral blood (PB) derived MSC migration and transmigration across endothelial cells. IL-17 increased PB-MSC migration in a wound healing assay as well as cell mobilization from collagen gel. Concomitantly IL-17 induced the expression of urokinase type plasminogen activator (uPA) without affecting matrix metalloproteinase expression. The incremented uPA expression mediated the capacity of IL-17 to enhance PB-MSC migration in a ERK1,2 MAPK dependent way. Also, IL-17 induced PB-MSC migration alongside with changes in cell polarization and uPA localization in cell protrusions. Moreover, IL-17 increased PB-MSC adhesion to endothelial cells and transendothelial migration, as well as increased the capacity of PB-MSC adhesion to fibronectin, in an uPA-dependent fashion. Therefore, our data suggested that IL-17 may act as chemotropic factor for PB-MSCs by incrementing cell motility and uPA expression during inflammation development.

Characteristics of human adipose mesenchymal stem cells isolated from healthy and cancer affected people and their interactions with human breast cancer cell line MCF‐7 in vitro

Adipose tissue is an attractive source of mesenchymal stem/stromal cells (MSCs) with potential applications in reconstructive plastic surgery and regenerative medicine. The aim of this study was to characterise human adipose tissue MSCs (ASCs) derived from healthy individuals and cancer patients and to compare their interactions with tumour cells. ASCs were isolated from adipose tissue of healthy donors, breast cancer‐adjacent adipose tissue of breast cancer patients and tumour‐adjacent adipose tissue of non‐breast cancer patients. Their proliferation, differentiation, immunophenotype and gene expression were assessed and effects on the proliferation of human breast cancer cell line MCF‐7 compared. ASCs from all sources exhibited similar morphology, proliferative and differentiation potential, showing the characteristic pattern of mesenchymal surface markers expression (CD90, CD105, CD44H, CD73) and the lack of HLA‐DR and hematopoietic markers (CD11a, CD33, CD45, Glycophorin‐CD235a), but uneven expression of CD34. ASCs also shared a common positive gene expression of HLA‐DR, HLA‐A, IL‐6, TGF‐β and HIF‐1, but were negative for HLA‐G, while the expression levels of Cox‐2 and IDO‐1 varied. All ASCs significantly stimulated the proliferation of MCF‐7 tumour cells in direct mixed co‐cultures and transwell system, although their conditioned media displayed antiproliferative activity. Data obtained showed that ASCs with similar characteristics are easily isolated from various donors and sites of origin, although ASCs could both suppress and favour tumour cells growth, emphasising the importance of cellular context within the microenvironment and pointing to the significance of safety studies to exclude any potential clinical risk of their application in regenerative medicine.

Inflammatory cytokines prime adipose tissue mesenchymal stem cells to enhance malignancy of MCF-7 breast cancer cells via transforming growth factor-β1.

Mesenchymal stem cells from human adipose tissue (hASCs) are proposed as suitable tools for soft tissue engineering and reconstruction. Although it is known that hASCs have the ability to home to sites of inflammation and tumor niche, the role of inflammatory cytokines in the hASCs‐affected tumor development is not understood. We found that interferon‐γ (IFN‐γ) and/or tumor necrosis factor‐α (TNF‐α) prime hASCs to produce soluble factors which enhance MCF‐7 cell line malignancy in vitro. IFN‐γ and/or TNF‐α‐primed hASCs produced conditioned media (CM) which induced epithelial to mesenchymal transition (EMT) of MCF‐7 cells by reducing E‐Cadherin and increasing Vimentin expression. Induced EMT was accompanied by increased invasion, migration, and urokinase type‐plasminogen activator (uPA) expression in MCF‐7 cells. These effects were mediated by increased expression of transforming growth factor‐β1(TGF‐β1) in cytokines‐primed hASCs, since inhibition of type I TGF‐β1 receptor on MCF‐7 cells and neutralization of TGF‐β1 disabled the CM from primed hASCs to increase EMT, cell migration, and uPA expression in MCF‐7 cells. Obtained data suggested that IFN‐γ and/or TNF‐α primed hASCs might enhance the malignancy of MCF‐7 cell line by inducing EMT, cell motility and uPA expression in these cells via TGF‐β1‐Smad3 signalization, with potentially important implications in breast cancer progression.

The Roles of Mesenchymal Stromal/Stem Cells in Tumor Microenvironment Associated with Inflammation

State of tumor microenvironment (TME) is closely linked to regulation of tumor growth and progression affecting the final outcome, refractoriness, and relapse of disease. Interactions of tumor, immune, and mesenchymal stromal/stem cells (MSCs) have been recognized as crucial for understanding tumorigenesis. Due to their outstanding features, stem cell-like properties, capacity to regulate immune response, and dynamic functional phenotype dependent on microenvironmental stimuli, MSCs have been perceived as important players in TME. Signals provided by tumor-associated chronic inflammation educate MSCs to alter their phenotype and immunomodulatory potential in favor of tumor-biased state of MSCs. Adjustment of phenotype to TME and acquisition of tumor-promoting ability by MSCs help tumor cells in maintenance of permissive TME and suppression of antitumor immune response. Potential utilization of MSCs in treatment of tumor is based on their inherent ability to home tumor tissue that makes them suitable delivery vehicles for immune-stimulating factors and vectors for targeted antitumor therapy. Here, we review data regarding intrusive effects of inflammatory TME on MSCs capacity to affect tumor development through modification of their phenotype and interactions with immune system.

Metabolic Plasticity of Stem Cells and Macrophages in Cancer

In addition to providing essential molecules for the overall function of cells, metabolism plays an important role in cell fate and can be affected by microenvironmental stimuli as well as cellular interactions. As a specific niche, tumor microenvironment (TME), consisting of different cell types including stromal/stem cells and immune cells, is characterized by distinct metabolic properties. This review will be focused on the metabolic plasticity of mesenchymal stromal/stem cells (MSC) and macrophages in TME, as well as on how the metabolic state of cancer stem cells (CSC), as key drivers of oncogenesis, affects their generation and persistence. Namely, heterogenic metabolic phenotypes of these cell populations, which include various levels of dependence on glycolysis or oxidative phosphorylation are closely linked to their complex roles in cancer progression. Besides well-known extrinsic factors, such as cytokines and growth factors, the differentiation and activation states of CSC, MSC, and macrophages are coordinated by metabolic reprogramming in TME. The significance of mutual metabolic interaction between tumor stroma and cancer cells in the immune evasion and persistence of CSC is currently under investigation.

The inhibition of periodontal ligament stem cells osteogenic differentiation by IL-17 is mediated via MAPKs.

Periodontal disease (PD), a degenerative bacterially induced disease of periodontium, can lead to bone resorption and teeth loss. Development of PD includes a strong inflammatory reaction, which involves multiple immune cells and their secreting factors including interleukin-17 (IL-17), which is not only an important modulator of immune and hematopoietic responses but also affects bone metabolism. In the present study we aimed to determine whether IL-17 affects the regenerative potential of periodontal ligament mesenchymal stem cells (PDLSCs) by investigating its ability to modulate osteogenic differentiation of these cells in vitro along with associated signaling pathways. Our results revealed that IL-17 inhibited both the proliferation and migration of PDLSCs and decreased their osteogenic differentiation by activating ERK1,2 and JNK mitogen-activated protein kinases. Obtained data suggested that IL-17 might contribute to alveolar bone loss in PD.

In vitro effects of IL-17 on angiogenic properties of endothelial cells in relation to oxygen levels

The aim of this study has been to elucidate how different oxygen levels impact the effects of Interleukin‐17 (IL‐17) on angiogenic properties of endothelial cells. Two endothelial cell lines, mouse MS‐1 and human EA.hy 926, were grown in 20% and 3% O2 and their angiogenic abilities analyzed after IL‐17 treatment: proliferation, apoptosis, migration and tubulogenesis. Expression of endothelial nitric oxide synthase (eNOS) and cyclooxygenase‐2 (Cox‐2) was also measured. Considering EA.hy 926 cell line, hypoxia alone reduced proliferation, survival and migration, but not their ability to form tubules. When cultured at 20% O2, IL‐17 stimulated proliferation, migration and tubulogenesis, whereas a hypoxic environment did not affect their migration and proliferation, but increased their survival and tubulogenic properties. Expression of eNOS and Cox‐2 increased by both IL‐17 and hypoxia, as well as with their combination. With the MS‐1 cell line hypoxia did not affect proliferation, survival, migration and tubule formation. At 20% O2, IL‐17 did not alter their proliferation,but inhibited migration and stimulated tubule formation. At 3% O2, only the stimulating effect of IL‐17 on tubulogenesis was evident. The constitutive expression of eNOS was unaffected by oxygen concentrations or IL‐17 supplementation, whereas both IL‐17 and hypoxia upregulated Cox‐2 expression. Thus the effects of IL‐17 on the angiogenic properties of endothelial cells depend on both the cell line used and the oxygen concentration.

Lipopolysaccharide can modify differentiation and immunomodulatory potential of periodontal ligament stem cells via ERK1,2 signaling

Lipopolysaccharide (LPS) is a pertinent deleterious factor in oral microenvironment for cells which are carriers of regenerative processes. The aim of this study was to investigate the emerging in vitro effects of LPS (Escherichia coli) on human periodontal ligament stem cell (PDLSC) functions and associated signaling pathways. We demonstrated that LPS did not affect immunophenotype, proliferation, viability, and cell cycle of PDLSCs. However, LPS modified lineage commitment of PDLSCs inhibiting osteogenesis by downregulating Runx2, ALP, and Ocn mRNA expression, while stimulating chondrogenesis and adipogenesis by upregulating Sox9 and PPARγ mRNA expression. LPS promoted myofibroblast‐like phenotype of PDLSCs, since it significantly enhanced PDLSC contractility, as well as protein and/or gene expression of TGF‐β, fibronectin (FN), α‐SMA, and NG2. LPS also increased protein and gene expression levels of anti‐inflammatory COX‐2 and pro‐inflammatory IL‐6 molecules in PDLSCs. Inhibition of peripheral blood mononuclear cells (MNCs) transendothelial migration in presence of LPS‐treated PDLSCs was accompanied by the reduction of CD29 expression within MNCs. However, LPS treatment did not change the inhibitory effect of PDLSCs on mitogen‐stimulated proliferation of CD4+ and the ratio of CD4+CD25high/CD4+CD25low lymphocytes. LPS‐treated PDLSCs did not change the frequency of CD34+ and CD45+ cells, but decreased the frequency of CD33+ and CD14+ myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity of MNCs, predominantly the CFU‐GM number. The results indicated that LPS‐activated ERK1,2 was at least partly involved in the observed effects on PDLSC differentiation capacity, acquisition of myofibroblastic attributes, and changes of their immunomodulatory features.