Comparative Analysis of Primary Ovarian Cancer Cells and Established Cell Lines as a New Tool for Studies on Ovarian Cancer Cell Complexity.

Primary cancer cells reflect the genetic background and phenotype of a tumor. Immortalized cells with higher proliferation activity have an advantage over primary cells. The aim of the study was to immortalize the primary ovarian cancer (OvCa) cells using the plasmid-carrying human telomerase reverse transcriptase (hTERT) gene and compare their phenotype and biological activity with the primary cells. The primary OvCa3 A and OvCa7 A cells were isolated from the ascitic fluid of two high-grade serous ovarian cancer patients and were characterized using immunocytochemical methods, flow cytometry, real-time RT-PCR, Western blot, metabolic activity, and migratory potential. Both immortalized ovarian cancer cell lines mirrored the phenotype of primary cancer cells, albeit with modifications. The OvCa3 A hTERT cells kept the mesenchymal stem cell phenotype of CD73/CD90/CD105-positivity and were CD133-negative, whereas the cell population of OvCa7 A hTERT lost CD73 expression, but almost 90% of cells expressed the CD133 characteristic for the CSCs phenotype. Immortalized OvCa cells differed in gene expression level with respect to Sox2 and Oct4, which was associated with stemness properties. The OvCa7 A hTERT cells showed higher metabolic and migratory activity and ALDH1 expression than the corresponding primary OvCa cells. Both primary and immortalized cell lines were able to form spheroids. The newly established unique immortalized cell line OvCa7 A hTERT, with the characteristic of a serous ovarian cancer malignancy feature, and with the accumulation of the p53, Pax8, and overexpression of the CD133 and CD44 molecules, may be a useful tool for research on therapeutic approaches, especially those targeting CSCs in ovarian cancer and in preclinical 2D and 3D models.

Feline umbilical cord mesenchymal stem cells: Isolation and in vitro characterization from distinct parts of the umbilical cord.

Mesenchymal stromal/stem cells (MSCs) are a particular population of cells that play an essential role in the regeneration potential of the body. As a source of MSCs, the umbilical cord (UC) has significant advantages, such as a no-risk procedure of tissue retrieval after birth and the easiness of MSCs isolation. In the presented study, the cells derived from the feline whole umbilical cord (WUC) and two separate parts of the UC tissue, including Wharton's jelly (WJ) and umbilical cord vessels (UCV), were investigated to check whether they exhibit MSCs characteristics. The cells were isolated and characterized based on their morphology, pluripotency, differentiation potential, and phenotype. In our study MSCs were successfully isolated and cultured from all UC parts; after one week of culture, the cells had a typical spindle shape consistent with MSCs morphology. Cells showed the ability to differentiate into chondrocytes, osteoblasts and adipocytes cells. Two markers typical of MSCs (CD44, CD90) and three pluripotency markers (Oct4, SOX2 and Nanog) were expressed in all cells cultures; but no expression of (CD34, MCH II) was evidenced by flow cytometry and RT-PCR. In addition, WJ-MSCs showed the highest ability of proliferation, more significant pluripotency gene expressions, and greater differentiation potential than the cells isolated from WUC and UCV. Finally, we conclude in this study that cat MSCs derived from all the parts are valuable cells that can be efficiently used in various fields of feline regenerative medicine, but cells from WJ can offer the best clinical utility.

"Endothelial Antibody Factory" at the Blood Brain Barrier: Novel Approach to Therapy of Neurodegenerative Diseases.

The failures of anti-ß-amyloid immunotherapies suggested that the very low fraction of injected antibodies reaching the brain parenchyma due to the filtering effect of the BBB may be a reason for the lack of therapeutic effect. However, there is no treatment, as yet, for the amyotrophic lateral sclerosis (ALS) despite substantial evidence existing of the involvement of TDP-43 protein in the evolution of ALS. To circumvent this filtering effect, we have developed a novel approach to facilitate the penetration of antibody fragments (Fabs) into the brain parenchyma. Leveraging the homing properties of endothelial progenitor cells (EPCs), we transfected, ex vivo, such cells with vectors encoding anti-ß-amyloid and anti-TDP43 Fabs turning them into an "antibody fragment factory". When injected these cells integrate into the BBB, where they secrete anti-TDP43 Fabs. The results showed the formation of tight junctions between the injected engineered EPCs and the unlabeled resident endothelial cells. When the EPCs were further modified to express the anti-TDP43 Fab, we could observe integration of these cells into the vasculature and the secretion of Fabs. Results confirm that production and secretion of Fabs at the BBB level leads to their migration to the brain parenchyma where they might exert a therapeutic effect.

Characterization of Biological Properties of Dental Pulp Stem Cells Grown on an Electrospun Poly(l-lactide-co-caprolactone) Scaffold.

Poly(l-lactide-co-caprolactone) (PLCL) electrospun scaffolds with seeded stem cells have drawn great interest in tissue engineering. This study investigated the biological behavior of human dental pulp stem cells (hDPSCs) grown on a hydrolytically-modified PLCL nanofiber scaffold. The hDPSCs were seeded on PLCL, and their biological features such as viability, proliferation, adhesion, population doubling time, the immunophenotype of hDPSCs and osteogenic differentiation capacity were evaluated on scaffolds. The results showed that the PLCL scaffold significantly supported hDPSC viability/proliferation. The hDPSCs adhesion rate and spreading onto PLCL increased with time of culture. hDPSCs were able to migrate inside the PLCL electrospun scaffold after 7 days of seeding. No differences in morphology and immunophenotype of hDPSCs grown on PLCL and in flasks were observed. The mRNA levels of bone-related genes and their proteins were significantly higher in hDPSCs after osteogenic differentiation on PLCL compared with undifferentiated hDPSCs on PLCL. These results showed that the mechanical properties of a modified PLCL mat provide an appropriate environment that supports hDPSCs attachment, proliferation, migration and their osteogenic differentiation on the PLCL scaffold. The good PLCL biocompatibility with dental pulp stem cells indicates that this mat may be applied in designing a bioactive hDPSCs/PLCL construct for bone tissue engineering.

From Primary MSC Culture of Adipose Tissue to Immortalized Cell Line Producing Cytokines for Potential Use in Regenerative Medicine Therapy or Immunotherapy.

For twenty-five years, attempts have been made to use MSCs in the treatment of various diseases due to their regenerative and immunomodulatory properties. However, the results are not satisfactory. Assuming that MSCs can be replaced in some therapies by the active factors they produce, the immortalized MSCs line was established from human adipose tissue (HATMSC1) to produce conditioned media and test its regenerative potential in vitro in terms of possible clinical application. The production of biologically active factors by primary MSCs was lower compared to the HATMSC1 cell line and several factors were produced only by the cell line. It has been shown that an HATMSC1-conditioned medium increases the proliferation of various cell types, augments the adhesion of cells and improves endothelial cell function. It was found that hypoxia during culture resulted in an augmentation in the pro-angiogenic factors production, such as VEGF, IL-8, Angiogenin and MCP-1. The immunomodulatory factors caused an increase in the production of GM-CSF, IL-5, IL-6, MCP-1, RANTES and IL-8. These data suggest that these factors, produced under different culture conditions, could be used for different medical conditions, such as in regenerative medicine, when an increased concentration of pro-angiogenic factors may be beneficial, or in inflammatory diseases with conditioned media with a high concentration of immunomodulatory factors.

HATMSC Secreted Factors in the Hydrogel as a Potential Treatment for Chronic Wounds-In Vitro Study.

Mesenchymal stem cells (MSCs) can improve chronic wound healing; however, recent studies suggest that the therapeutic effect of MSCs is mediated mainly through the growth factors and cytokines secreted by these cells, referred to as the MSC secretome. To overcome difficulties related to the translation of cell therapy into clinical use such as efficacy, safety and cost, we propose a hydrogel loaded with a secretome from the recently established human adipose tissue mesenchymal stem cell line (HATMSC2) as a potential treatment for chronic wounds. Biocompatibility and biological activity of hydrogel-released HATMSC2 supernatant were investigated in vitro by assessing the proliferation and metabolic activity of human fibroblast, endothelial cells and keratinocytes. Hydrogel degradation was measured using hydroxyproline assay while protein released from the hydrogel was assessed by interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) ELISAs. Pro-angiogenic activity of the developed treatment was assessed by tube formation assay while the presence of pro-angiogenic miRNAs in the HATMSC2 supernatant was investigated using real-time RT-PCR. The results demonstrated that the therapeutic effect of the HATMSC2-produced factors is maintained following incorporation into collagen hydrogel as confirmed by increased proliferation of skin-origin cells and improved angiogenic properties of endothelial cells. In addition, HATMSC2 supernatant revealed antimicrobial activity, and which therefore, in combination with the hydrogel has a potential to be used as advanced wound-healing dressing.

Suppression of Ovarian Cancer Cell Growth by AT-MSC Microvesicles.

Transport of bioactive cargo of microvesicles (MVs) into target cells can affect their fate and behavior and change their microenvironment. We assessed the effect of MVs derived from human immortalized mesenchymal stem cells of adipose tissue-origin (HATMSC2-MVs) on the biological activity of the ovarian cancer cell lines ES-2 (clear cell carcinoma) and OAW-42 (cystadenocarcinoma). The HATMSC2-MVs were characterized using dynamic light scattering (DLS), transmission electron microscopy, and flow cytometry. The anti-tumor properties of HATMSC2-MVs were assessed using MTT for metabolic activity and flow cytometry for cell survival, cell cycle progression, and phenotype. The secretion profile of ovarian cancer cells was evaluated with a protein antibody array. Both cell lines internalized HATMSC2-MVs, which was associated with a decreased metabolic activity of cancer cells. HATMSC2-MVs exerted a pro-apoptotic and/or necrotic effect on ES-2 and OAW-42 cells and increased the expression of anti-tumor factors in both cell lines compared to control. In conclusion, we confirmed an effective transfer of HATMSC2-MVs into ovarian cancer cells that resulted in the inhibition of cell proliferation via different pathways, apoptosis and/or necrosis, which, with high likelihood, is related to the presence of different anti-tumor factors secreted by the ES-2 and OAW-42 cells.

Microvesicles from Human Immortalized Cell Lines of Endothelial Progenitor Cells and Mesenchymal Stem/Stromal Cells of Adipose Tissue Origin as Carriers of Bioactive Factors Facilitating Angiogenesis.

Endothelial progenitor cells (EPCs) and mesenchymal stem/stromal cells (MSCs) are associated with maintaining tissue homeostasis and tissue repair. Both types of cells contribute to tissue regeneration through the secretion of trophic factors (alone or in the form of microvesicles). This study investigated the isolation and biological properties of microvesicles (MVs) derived from human immortalized MSC line HATMSC1 of adipose tissue origin and EPC line. The human immortalized cell line derived from the adipose tissue of a patient with venous stasis was established in our laboratory using the hTERT and pSV402 plasmids. The human EPC line originating from cord blood (HEPC-CB.1) was established in our previous studies. Microvesicles were isolated through a sequence of centrifugations. Analysis of the protein content of both populations of microvesicles, using the Membrane-Based Antibody Array and Milliplex ELISA showed that isolated microvesicles transported growth factors and pro- and antiangiogenic factors. Analysis of the miRNA content of isolated microvesicles revealed the presence of proangiogenic miRNA (miR-126, miR-296, miR-378, and miR-210) and low expression of antiangiogenic miRNA (miR-221, miR-222, and miR-92a) using real-time RT-PCR with the TaqMan technique. The isolated microvesicles were assessed for their effect on the proliferation and proangiogenic properties of cells involved in tissue repair. It was shown that both HEPC-CB.1- and HATMSC1-derived microvesicles increased the proliferation of human endothelial cells of dermal origin and that this effect was dose-dependent. In contrast, microvesicles had a limited impact on the proliferation of fibroblasts and keratinocytes. Both types of microvesicles improved the proangiogenic properties of human dermal endothelial cells, and this effect was also dose-dependent, as shown in the Matrigel assay. These results confirm the hypothesis that microvesicles of HEPC-CB.1 and HATMSC1 origin carry proteins and miRNAs that support and facilitate angiogenic processes that are important for cutaneous tissue regeneration.

Activity of the human immortalized endothelial progenitor cell line HEPC-CB.1 supporting in vitro angiogenesis.

The human HEPC-CB.1 cell line with many characteristics of endothelial progenitor cells (EPC) was tested for its proangiogenic properties as a potentially therapeutic compound. HEPC-CB.1 cells' potential to differentiate into endothelial cells was revealed after treating the cells with a mixture of ATRA, cAMP and VEGF, as shown by the reduced expression levels of CD133, CD271 and CD90 antigens, augmentation of CD146 and CD31, and a decrease in cell clonogenicity. The cooperation of HEPC-CB.1 with the endothelial cell line HSkMEC.2 resulted in the formation of a common network. Tube formation was significantly more effective when resulting from HEPC-CB.1 and HSkMEC.2 cell co-culture as compared to a monoculture of each cell line. The exocrine mechanism of HEPC-CB.1 and HSkMEC.2 cross talk by secreted factors was evidenced using the HEPC-CB.1 supernatant to increase the efficacy of HSkMEC.2 tube formation. The proangiogenic factors produced by HEPC-CB.1 were identified using cytokine antibody array. Out of 120 examined factors, the HEPC-CB.1 cell line produced 63, some with known angiogenic activity. As in vivo the angiogenic process occurs at low oxygen tension, it was observed that in hypoxia, the production of defined factors was augmented. The presented results demonstrate that HEPC-CB.1 cells are able to both cooperate and integrate in a newly formed network and produce factors that help the network formation. The results suggest that HEPC-CB.1 cells are indeed endothelial progenitors and may prove to be an effective tool in regenerative medicine.

Autotransplantation of the Adipose Tissue-Derived Mesenchymal Stromal Cells in Therapy of Venous Stasis Ulcers.

Adipose tissue is a reliable source of mesenchymal stromal cells (MSC) for use in regenerative medicine. The aim of this pilot study was to describe the method, and assess the safety and the potential efficacy of transplantation of autologous adipose tissue-derived MSC for the treatment of chronic venous stasis ulcers. Study group consisted of 11 patients (mean age: 66.6 ± 9.5 years) with chronic venous stasis ulcers. Adipose tissue was harvested by tumescent-aspiration method. Stromal cells were separated using a dedicated closed system in a real-time bedside manner. The phenotype of cells was determined immediately after separation. Cell concentrate was implanted subcutaneously around the wound and the wound bed. All ulcers were assessed planimetrically before autotransplantation and every two weeks during the six-month follow-up. During the study all patients received standard local and general treatment. The preparation contained an average of 5.6 × 106 ± 4 × 106 cells per milliliter. The phenotype of 65-82% of transplanted cells expressed MSC markers: CD73+ CD90+ and CD34+. An improvement was observed in 75% of ulcers. The data showed highly significant negative correlation (p < 0.0001) between wound size and wound closure degree. There was no correlation of ulcer healing with other parameters evaluated, including age of the patients. No serious side effects were observed. Autotransplantation of adipose tissue stromal cells may be a safe and promising treatment method for chronic venous ulcers.

Can supernatant from immortalized adipose tissue MSC replace cell therapy? An in vitro study in chronic wounds model.

BACKGROUND: Mesenchymal stem cells (MSCs) secrete a cocktail of growth factors and cytokines, which could promote tissue regeneration and wound healing. Therefore, in clinical practice, post-culture MSC supernatant treatment could be a more attractive alternative to autologous stem cell transplantation. In this study, we compared the regenerative properties of supernatants harvested from four newly established human adipose tissue mesenchymal stem cell lines (HATMSCs) derived from chronic wound patients or healthy donors. METHODS: HATMSC supernatants were produced in a serum-free medium under hypoxia and their content was analyzed by a human angiogenesis antibody array. The regenerative effect of HATMSCs supernatants was investigated in an in vitro model of chronic wound, where cells originating from human skin, such as microvascular endothelial cells (HSkMEC.2), keratinocytes (HaCaT), and fibroblasts (MSU-1.1), were cultured in serum-free and oxygen-reduced conditions. The effect of supernatant treatment was evaluated using an MTT assay and light microscopy. In addition, fibroblasts and HATMSCs were labeled with PKH67 and PKH26 dye, respectively, and the effect of supernatant treatment was compared to that obtained when fibroblasts and HATMSCs were co-cultured, using flow cytometry and fluorescent microscopy. RESULTS: A wide panel of angiogenesis-associated cytokines such as angiogenin, growth-regulated oncogene (GRO), interleukin-6 and 8 (IL-6, IL-8), vascular endothelial growth factor (VEGF), insulin growth factor 1 (IGF-1), and matrix metalloproteinase (MMP) were found in all tested HATMSCs supernatants. Moreover, supernatant treatment significantly enhanced the survival of fibroblasts, endothelial cells, and keratinocytes in our chronic wound model in vitro. Importantly, we have shown that in in vitro settings, HATMSC supernatant treatment results in superior fibroblast proliferation than in the case of co-culture with HATMSCs. CONCLUSIONS: Our results suggest that therapy based on bioactive factors released by the immortalized atMSC into supernatant has important effect on skin-derived cell proliferation and might preclude the need for a more expensive and difficult cell therapy approach to improve chronic wound healing.

Functionalization with a VEGFR2-binding antibody fragment leads to enhanced endothelialization of a cardiovascular stent in vitro and in vivo.

Rapid endothelialization of cardiovascular stents is critical to prevent major clinical complications such as restenosis. Reconstruction of the native endothelium on the stent surface can be achieved by the capture of endothelial progenitor cells (EPCs) or neighboring endothelial cells (ECs) in vivo. In this study, stainless steel cardiovascular stents were functionalized with recombinant scFv antibody fragments specific for vascular endothelial growth factor receptor-2 (VEGFR2) that is expressed on EPCs and ECs. Anti-VEGFR2 scFvs were expressed in glycosylated form in Escherichia coli and covalently attached to amine-functionalized, titania-coated steel disks and stents. ScFv-coated surfaces exhibited no detectable cytotoxicity to human ECs or erythrocytes in vitro and bound 15 times more VEGFR2-positive human umbilical vein ECs than controls after as little as 3 min. Porcine coronary arteries were successfully stented with scFv-coated stents with no adverse clinical events after 30 days. Endovascular imaging and histology revealed coverage of the anti-VEGFR2 scFv-coated stent with a cell layer after 5 days and the presence of a neointima layer with a minimum thickness of 80 µm after 30 days. Biofunctionalization of cardiovascular stents with endothelial cell-capturing antibody fragments in this manner offers promise in accelerating stent endothelialization in vivo. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:213-224, 2020.

Metallacarboranes as a tool for enhancing the activity of therapeutic peptides.

Metallacarboranes are anionic boron clusters with high affinity to serum albumin, ability to cross biological membranes, and no apparent toxicity in vitro and in vivo. Thus, conjugation with cobalt bis(1,2-dicarbollide), [COSAN]- , ([3,3'-Co(1,2-C2 B9 H11 )2 ]- ) may improve the properties of therapeutic peptides or proteins at both molecular and systemic levels. Here, we conjugated [COSAN]- with the therapeutic peptide thymosin ß4 (Tß4), which has a pleiotropic activity that results in enhanced healing and regeneration of injured tissues. Using fluorescence quenching of human serum albumin and surface plasmon resonance techniques, we showed that the conjugates have a high affinity to human serum albumin. Using an in vitro wound closure assay, we showed that conjugation with [COSAN]- enhances the activity of Tß4 toward fibroblasts (MSU1.1 cell line). These results indicate an application of metallacarboranes in the development of analogs of various therapeutic peptides/proteins with superior pharmacological properties.

MCM5 Expression Is Associated With the Grade of Malignancy and Ki-67 Antigen in LSCC.

BACKGROUND/AIM: The minichromosome maintenance proteins (MCMs) may be potential biomarkers of cancer cell proliferation. They are essential to initiate DNA replication. The aim of the study was to investigate the level of MCM5 expression in benign lesions (BLs) and laryngeal squamous cell cancer (LSCC). MATERIALS AND METHODS: Immunohistochemical (IHC) analysis was carried out on 83 LSCCs and 10 BLs. Western-blot, immunofluorescence analysis (IF) and real-time PCR (RT-PCR) were performed using HEp-2 cancer cells and HaCaT keratinocytes. RESULTS: The expression of MCM5 was higher in LSCC than in the BLs (p<0.0001) and was higher in subsequent malignancies of LSCC. Positive correlations were demonstrated between the expression levels of MCM5 and the Ki-67 antigen. In vitro studies have confirmed that the expression of MCM5 is elevated in cancer cells. CONCLUSION: MCM5 protein may be used as a potential marker of cancer cell proliferation in LSCC.

UCHL1/PGP 9.5 Dynamic in Neuro-Immune-Cutaneous Milieu: Focusing on Axonal Nerve Terminals and Epidermal Keratinocytes in Psoriatic Itch.

Psoriasis is an immunogenetic skin disease manifesting as plaque lesions on the skin. Patients with psoriasis frequently suffer from itch, an unpleasant sensation causing a desire to scratch. Psoriatic itch is mainly transmitted by unmyelinated C-fibers; however, the exact molecular mechanism of psoriatic itch is still unexplained. Protein gene product 9.5 (PGP 9.5) is a panneurological marker commonly used for analysis of peripheral peptidergic and nonpeptidergic nerves and identification of cutaneous neuro-immune-endocrine cells. However, some studies suggested that nonneuronal cells, like keratinocytes, may also express PGP 9.5. This phenomenon might be linked with impaired axonal transport, keratinocyte injury, or dysfunctions of neuro-immune-cutaneous connections. The aim of this study was to analyze the expression of PGP 9.5 in psoriatic skin. We observed significantly altered density of PGP 9.5-positive axonal nerve terminals in pruritic lesional (p=0.04) and nonlesional psoriatic skin (p>0.001) compared with controls. In contrast, no significant differences were observed between psoriatic skin without itch and controls. Furthermore, PGP 9.5 expression by suprabasal keratinocytes (SBKs) was significantly increased in itchy skin lesions (p=0.007) compared to skin without itch, and a positive correlation was observed between PGP 9.5 expression and itch intensity (r=0.64; p=0.02). Our findings indicate changes in peripheral innervations and psoriatic keratinocytes, which may influence neuro-immune-cutaneous homeostasis and modulate itch transmission.

MRP1 protein expression in leukemic stem cells as a negative prognostic marker in acute myeloid leukemia patients.

BACKGROUND: It is well established that expression of multi-drug resistance (MDR) proteins (MDR1, BCRP, MDR3, MRP1, and LRP) in leukemic blasts correlates with acute myeloid leukemia (AML) patients' clinical response. Assuming that leukemic stem cells (LSC) are resistant to chemotherapy and responsible for relapse, it might be clinically relevant to evaluate the expression level of MDR proteins in LSC and relate it to the clinical outcome. METHODS: Bone marrow samples from 26 patients with de novo AML were labeled with antibodies to distinguish CD34+CD38-CD123+ LSC population and with antibodies against MDR1, BCRP, MDR3, MRP1, or LRP proteins. Multicolor flow cytometry was applied to evaluate the expression of MDR proteins in blasts and LSC. RESULTS: Nine of 26 patients with AML attained CR (30%). High negative correlation was found between MDR1 and LRP expression in blasts and the patient's remission. MDR proteins were expressed more frequently in LSC than in leukemic blasts. High negative correlation was also observed between remission achievement and MRP1 expression in LSC. CONCLUSIONS: Our data present for the very first time the high negative correlation between MRP1 protein expression in LSC and AML patients' remission. It does strongly suggest that MRP1 expression in LSC is an adverse prognostic marker in patients with de novo AML.

Nestin-positive microvessel density is an independent prognostic factor in breast cancer.

The process of angiogenesis based on new vessel formation within the tumour area plays a significant role in the progression of breast cancer. Nestin is an intermediate filament protein and participates in the cytoskeleton organization. Nestin expression in the endothelium of blood vessels is mainly limited to newly forming vessels, thus being a more specific marker of angiogenesis than the commonly used vascular antigens. The aim of this study was to determine the prognostic value of nestin-positive microvessel density (Nes+MVD) in breast cancer patients and to confirm that nestin expression is related to newly forming tumour vessels. In this study, 137 cases of ductal breast carcinoma and 19 cases of non-malignant breast tissue lesions (NBTLs) were examined. Immunohistochemical reactions were performed on paraffin sections using antibodies against nestin, CD34 and CD31 antigens. For each marker, the microvessel density (MVD) was determined. Nestin expression was also examined in human endothelial cell lines (HUVEC-SVT, HMEC-1 and HEPC-CB.1) representing a different level of endothelial cell maturity. HUVEC-SVT and HMEC-1 cells represent the endothelium of mature vessels, whereas HEPC-CB.1 cells represent the early endothelial progenitor cells (EPCs). We have demonstrated that high Nes+MVD may be associated with a more aggressive course of the disease and a poorer prognosis. We have also found a higher Nes+MVD in the cases with lymph node metastases, with higher histological grade, with advanced-stage disease and with the triple-negative (TN) breast cancer. In addition, nestin expression in vessels was associated with a shorter overall survival (OS) and earlier relapse, and in the case of OS nestin was an independent prognostic factor. Finally, we further confirmed that nestin expression in endothelial cells reflects a progenitor nature of newly forming vessels.

Increased Endothelial Progenitor Cell Number in Early Stage of Endometrial Cancer.

OBJECTIVES: It is generally believed that circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) reflect the state of the endothelium, its injury and/or repair possibilities. In different types of cancers, increased numbers of CECs and EPCs were found, suggesting their participation in cancer angiogenesis. The objective of this study was to determine whether, in the blood circulation of women with early endometrial cancer, CEC and EPC levels differ from those of healthy women of similar age. METHODS: For CEC number evaluation, samples of peripheral blood cells of women with endometrial carcinoma and control subjects were labeled with anti-CD31 and anti-CD45 antibodies; for EPCs, with anti-VEGFR2 (vascular-endothelium growth factor receptor 2)/KDR and anti-CD34 antibodies. The CEC and EPC cells were then quantified by flow cytometry. RESULTS: Endothelial progenitor cell numbers (CD34, VEGFR2/KDR) in the peripheral blood of women with endometrial carcinoma were significantly augmented as compared with those of control healthy women and CEC numbers (CD31, CD45) were similar in both groups. Cancer patients were divided according to the grading into G1 and G2 groups and according to the stage into International Federation of Gynecology and Obstetrics (FIGO) stage IA and FIGO IB groups. Statistically significant augmented EPC numbers were demonstrated only in G1 and FIGO IA patients. CONCLUSIONS: These results strongly suggest new vessel formation from recruited endothelial precursors as being involved mainly at the early stages of tumor progression.

A 3D model of tumour angiogenic microenvironment to monitor hypoxia effects on cell interactions and cancer stem cell selection.

Tumour microenvironment determines the fate of treatments. Reconstitution of tumour conditions is mandatory for alternative in vitro methods devoted to cancer development and the selection of therapeutic strategies. This work describes a 3D model of melanoma growth in its environment. Introducing means to mimic tumour angiogenesis, which turns on tumour progression, the model shows that melanoma tumour spheroids allow reconstitution of solid tumours with stromal cells. Angiogenesis evidenced the differential recruitment of endothelial cells (EC) from early progenitors (EEPCs) to mature ECs. Hypoxia was the key parameter that selected and stabilized melanoma cancer stem like cells (CSCs) phenotype based on aldehyde dehydrogenase expression as the best criterion. The 3D-tumour-model demonstrated the distinct reactivity of ECs toward tumour cells in terms of cellular cross-talk and humoral response. Intra-spheroid cell-to-cell membrane dye exchanges, mediated by intercellular interactions, uncovered the melanoma-to-EEPC cooperation. The resulting changes in tumour milieu were evidenced by the chemokinic composition and hypoxia-related variations in microRNA expression assessed in each cellular component of the spheroids. This method brings new tools to decipher the molecular mechanism of tumour-mediated cell recruitment and for in vitro assessment of therapeutic approaches.

Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation.

Active cellular transporters of harmful agents-multidrug resistance (mdr) proteins-are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins-MDR1, BCRP, MRP1, MRP4 and MRP5-in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific.