Ability Of Conditioned Medium Research Articles

Hypoxia-induced polarization of M2 macrophages and C-C motif chemokine ligand 5 secretion promotes the migration and invasion of trophoblasts†.

In the early stage of pregnancy, hypoxia in the placenta is of great significance to the migration and invasion of trophoblasts. In addition, changes to the polarity and activity of macrophages can affect embryo implantation, trophoblast migration and invasion, and vascular remodeling by affecting cytokine secretion. However, the mechanism of the effects of hypoxic conditions in the placenta on trophoblasts remains unknown. We used gene knockdown on macrophages, and drug treatment on trophoblasts, and cultured them under hypoxic and normoxic conditions. The cells were then subjected to wound-healing assays, Transwell cell invasion experiments, quantitative real-time reverse transcription Polymerase Chain Reaction (PCR), western blotting, and immunofluorescence. The polarization of macrophages in each group, the migration and invasion ability of trophoblasts, and changes to the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway were detected. Hypoxic conditions induce M2 polarization of macrophages. The conditioned medium from macrophages under hypoxic conditions increased the migration and invasion of trophoblasts and enhanced the levels of phosphorylated (p)-PI3K and p-AKT in trophoblasts. After C-C motif chemokine ligand 5 knockdown in macrophages, the ability of conditioned medium from macrophages cultured under hypoxic conditions to promote the migration and invasion of trophoblasts was weakened significantly. The use of PI3K/AKT signaling pathway agonists could reverse the attenuation effect caused by C-C motif chemokine ligand 5 knockdown.

Conditioned media from mesenchymal stromal cells and periodontal ligament fibroblasts under cyclic stretch stimulation promote bone healing in mouse calvarial defects

Background aimsWhen cells are exposed to stresses such as mechanical stimuli, they release growth factors and adapt to the surrounding environment H ere, we demonstrated that mechanical stimulation during culture affects the production of osteogenic and angiogenic factors. MethodsHuman bone marrow derived mesenchymal stromal cells (hMSCs) and human periodontal ligament fibroblasts (HPLFs ) were cultured under cyclic stretch stimulation for 24 h. Collected of the cells and conditioned media (CM), the gene and protein expression levels of osteogenic and angiogenic factors were evaluated. CM was also evaluated for angiogenic activity and calc ification ability. In in vivo study, CM was administered to a mouse calvarial defect model and histologically and radiologically evaluated. ResultsQuantitative real time polymerase chain reaction results showed that the expression of bone morphogenetic pro tein 2, 4 (BMP 2, 4), vascular endothelial growth factor A (VEGF A), and platelet derived growth factor AA (PDGF AA) was upregulated in the cyclic stretch stimulation group in comparison with the non stretch group in each cell type. Enzyme linked immunosor bent assay results revealed that the expression of BMP 2,4, VEGF A was upregulated in the cyclic stretch group in comparison with the non stretch group in each cell type. Only HPLFs showed significant difference in PDGF AA expression between the cyclic str etch and the non stretch group. Tube formation assay and Alizarin Red S staining results showed that angiogenic activity and calcification ability of CM was upregulated in the cyclic stretch stimulation group in comparison with the non stretch group in eac h cell type. CM was administered to the mouse calvarial defect model. Histological and radiological examination showed that the bone healing was promoted by CM from the cyclic stretch culture group. Immunohistological staining revealed that CM from cyclic stretch group have greater angiogenic effect than CM from the non stretch group. ConclusionsThese results indicate that osteogenesis was promoted by CM obtained under cyclic stretch stimulation through the increase of angiogenesis in the mouse calvarial defect model.

The Ability of Conditioned Media From Stem Cells to Repair Vocal Fold Injuries.

This study investigated the ability of hypoxia-induced 25-fold concentrated conditioned media (hCM) from human nasal inferior turbinate-derived mesenchymal stem cells (hTMSC) to repair injured vocal folds during the early phase of the wound-healing process. The vocal fold was injured in Sprague-Dawley rats. Next, hCM from hTMSC (the hCM group) or hTMSC (the hTMSC group) were injected into the injured vocal folds. As a control, saline (the phosphate-buffered saline group) or 25-fold concentrated media (the media group) was injected in the same manner. The vocal folds were harvested for quantitative real-time polymerase chain reaction (PCR) at 1 week and 2 weeks after injury. Histologic evaluation was performed at 3 weeks postinjury. In the hCM group at 1 week after injury, PCR showed that the genes encoding hyaluronan synthase (HAS), HAS 1, and HAS 2 were significantly upregulated compared to the media and normal groups. The gene encoding procollagen III was significantly downregulated compared to the media group. Nearly identical results were obtained for the hTMSC group at 1 week after injury. Histological examination showed that the hCM group was similar to or better than the hTMSC group in collagen deposition and hyaluronic acid production. The injection of hCM into injured vocal folds produced antifibrotic effects in the early phase of wound healing. These effects were equivalent to those produced by the injection of hTMSC. These results provide a foundation for the future clinical use of hCM for vocal fold regeneration. NA Laryngoscope, 129:1867-1875, 2019.

Abstract 206: Electrical Stimulation Enhances the Therapeutic Potential of Pediatric Cardiac Progenitor Cells

Nearly 1 in every 120 children born has a congenital heart defect, and many of these children go on to develop heart failure (HF). The emergence of cardiovascular regenerative medicine as a potential therapeutic strategy for pediatric HF has provided new avenues for treatment. Stem cells such as cardiac progenitor cells (CPCs) have shown promise in restoring cardiac function in adult pathologies; however, few studies have focused on the pediatric population. Recently, pediatric CPCs >1year old have been shown to have reduced efficacy compared to younger cells. This indicates a need to enhance the regenerative potential of this population of cells for an autologous treatment for HF in pediatric patients. The regenerative potential of adult stem cells can be enhanced by ex vivo manipulation, and electrical stimulation (ES) is one such treatment we have examined in pediatric CPCs. We have previously shown that pediatric CPCs (1-5 years old) respond to ES by initiating intracellular Ca 2+ oscillations, and this induces phenotypic and genotypic changes in these cells. We then sought to examine the regenerative capacity of pediatric CPCs after conditioning with ES. In this study, we characterized the ES-induced changes in pediatric CPCs and showed they had a positive effect on the regenerative potential of these cells. ES-treated CPCs resulted in improved RV function following delivery compared with untreated CPCs in a juvenile rat model of right ventricular HF. Analysis of Ca 2+ handling in CPCs after chronic ES showed that these cells do not develop Ca 2+ transients or contractility similar to adult myocytes, and likely do not directly contribute to the contractility of the heart after injection. To determine if the effects were due to paracrine signaling, we tested the ability of conditioned media from these cells to regulate the function of failing cardiac myocytes. Isolated failing myocytes treated with conditioned media from CPCs showed improved Ca 2+ handling and contractility compared to untreated controls. Our data suggest that ES represents a novel treatment to enhance the therapeutic potential of pediatric CPCs, and the means of action of the modified cells is in part through a paracrine effect on resident cardiomyocytes.

Secretome of Differentiated PC12 Cells Enhances Neuronal Differentiation in Human Mesenchymal Stem Cells Via NGF-Like Mechanism

The secretome-mediated responses over cellular physiology are well documented. Stem cells have been ruling the field of secretomics and its role in regenerative medicine since the past few years. However, the mechanistic aspects of secretome-mediated responses and the role of other cells in this area remain somewhat elusive. Here, we investigate the effects of secretome-enriched conditioned medium (CM) of neuronally differentiated PC12 cells on the neuronal differentiation of human mesenchymal stem cells (hMSCs). The exposure to CM at a ratio of 1:1 (CM: conditioned medium of PC12 cells) led to neuronal induction in hMSCs. This neuronal induction was compared with a parallel group of cells exposed to nerve growth factor (NGF). There was a marked increase in neurite length and expression of neuronal markers (β-III tubulin, neurofilament-M (NF-M), synaptophysin, NeuN in exposed hMSCs). Experimental group co-exposed to NGF and CM showed an additive response via MAPK signaling and directed the cells particularly towards cholinergic lineage. The ability of CM to enhance the neuronal properties of stem cells could aid in their rapid differentiation into neuronal subtypes in case of stem cell transplantation for neuronal injuries, thus broadening the scope of non-stem cell-based applications in the area of secretomics.

Abstract 1139: G protein-coupled receptors (GPCRs): unrecognized potential therapeutic targets in cancer

Abstract G protein-coupled receptors (GPCRs) are the largest class of signaling receptors in humans and other species and in addition, the most widely targeted class for FDA-approved therapeutics. However, GPCRs are not commonly targeted in cancers other than endocrine tumors. We hypothesize that GPCRs may have been “missed” as targets in cancer, perhaps in part because they have not been frequently identified as driver mutations; however, GPCRs may have unappreciated utility as therapeutic targets in a variety of tumors. To test this hypothesis, we have used unbiased approaches (Taqman GPCR arrays and RNA-seq), mining of publicly available datasets (e.g., The Cancer Genome Atlas, TCGA), and validation studies (e.g., protein and functional analyses) to assess GPCRs in a variety of human tumors, cancer cells and stromal cells (cancer-associated fibroblasts, CAFs). A major focus of our efforts has been on pancreatic ductal adenocarcinoma (PDAC) cells/tumors and pancreatic CAFs (PCAFs). We find that PDAC cells and PCAFs express >70 different GPCRs, including many orphan GPCRs (receptors without known physiologic agonists) and that numerous GPCRs are expressed at much higher levels than in normal precursor cells (pancreatic ductal epithelial cells for PDAC cells and pancreatic stellate cells and pancreatic fibroblasts for PCAFs). We find that a cluster of GPCRs is overexpressed in PDAC tumors. Two such receptors are orphan GPCRs, Orphan 1 and Orphan A, neither of which is mutated but each has high expression, respectively, in PCAFs and PDAC cells. Orphan 1 and Orphan A have at least 2-fold higher expression in >90% of PDAC tumors in TCGA compared to that in normal pancreatic tissue (compiled from the GTEX database). Higher expression of orphan A is associated with decreased survival and remission, while Orphan 1 expression correlates with that of numerous fibrotic genes. Importantly, both Orphan 1 and Orphan A are functional and appear to contribute to the malignant phenotype. For example, siRNA knockdown of Orphan 1 in PCAFs blunts production of profibrotic markers and decreases ability of conditioned media from PCAFs to enhance proliferation of PDAC cells while transfection of Orphan A increases DNA synthesis of normal pancreatic ductal epithelial cells. Taken together, these and other results suggest that in addition to their role in endocrine tumors, GPCRs represent previously unrecognized contributors to cancer through actions on tumor cells and stromal cells. These data suggest the possibility that highly expressed GPCRs (such as Orphan A) may function as oncogenes. Thus, GPCRs may be druggable, novel targets for the treatment of cancer, including pancreatic cancer, a tumor for which new therapeutics are an important, unmet need. Note: This abstract was not presented at the meeting. Citation Format: Paul A. Insel, Krishna Sriram, Shu Z. Wiley, Thalia McCann, Randall P. French, Andrew M. Lowy. G protein-coupled receptors (GPCRs): unrecognized potential therapeutic targets in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1139. doi:10.1158/1538-7445.AM2017-1139

MDA-MET-conditioned-medium augments the chemoattractant-dependent migration of MDA-MET cells towards hFOB-conditioned medium and increases collagenase activity

BackgroundMetastasis of breast cancer displays site-specificity towards bone. Recently, studies have emerged indicating that primary tumors may remotely influence creation of a pre-metastatic niche. In this study, we used human fetal osteoblastic cells and MDA-MET, a metastatic and preferentially bone homing derivative of the breast cancer cell line MDA-MB-231. We examined 1) whether secreted factors from MDA-MET cells increase generation of chemoattractants by human foetal osteoblastic cells 2) whether MDA-MET cells were responsive to these chemoattractants and 3) the identity of these chemoattractants.MethodsHuman foetal osteoblastic cells were treated with conditioned medium of MDA-MET cells for 24 hours and then washed with phosphate-buffered saline. Serum-free replacement medium was conditioned by treated hFOB cells for 18 hours, before its use in in vitro quantification of MDA-MET migration. We also quantified collagen levels and collagenase activity in conditioned medium from human foetal osteoblastic cells.ResultsConditioned medium from human foetal osteoblastic cells that had been treated with MDA-MET-conditioned medium attracted more MDA-MET cells than hFOB cells pre-exposed to their own medium. This conditioned medium had increased collagenase activity. The addition of bacterial collagenase removed the ability of conditioned medium from human foetal osteoblastic cells to attract MDA-MET cells.ConclusionsOur data suggest that an increase in collagenase activity in osteoblastic cells induced by their exposure to breast cancer cell–secreted factors may increase their ability to attract breast cancer cells.

Severe Convulsions and Dysmyelination in Both Jimpy and Cx32/47 -/- Mice may Associate Astrocytic L-Channel Function with Myelination and Oligodendrocytic Connexins with Internodal Kv Channels.

The Jimpy mouse illustrates the importance of interactions between astrocytes and oligodendrocytes. It has a mutation in Plp coding for proteolipid protein and DM20. Its behavior is normal at birth but from the age of ~2weeks it shows severe convulsions associated with oligodendrocyte/myelination deficits and early death. A normally occurring increase in oxygen consumption by highly elevated K+ concentrations is absent in Jimpy brain slices and cultured astrocytes, reflecting that Plp at early embryonic stages affects common precursors as also shown by the ability of conditioned medium from normal astrocytes to counteract histological abnormalities. This metabolic response is now known to reflect opening of L-channels for Ca2+. The resulting deficiency in Ca2+ entry has many consequences, including lack of K+-stimulated glycogenolysis and release of gliotransmitter ATP. Lack of purinergic stimulation compromises oligodendrocyte survival and myelination and affects connexins and K+ channels. Mice lacking the oligodendrocytic connexins Cx32 and 47 show similar neurological dysfunction as Jimpy. This possibly reflects that K+ released by intermodal axonal Kv channels is transported underneath a loosened myelin sheath instead of reaching the extracellular space via connexin-mediated transport to oligodendrocytes, followed by release and astrocytic Na+,K+-ATPase-driven uptake with subsequent Kir4.1-facilitated release and neuronal uptake.

The roles of supernatant of macrophage treated by excretory-secretory products from muscle larvae of Trichinella spiralis on the differentiation of C2C12 myoblasts

The excretory-secretory products (ESPs) released by the muscle-larvae (ML) stage of Trichinella spiralis have been suggested to be involved in nurse cell formation. However, the molecular mechanisms by which ML-ESPs modulate nurse cell formation remain unclear. Macrophages exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for skeletal muscle repair, notably, via their actions on myogenic precursor cells. However, these interactions during T. spiralis infection have not been characterized. In the present study, the ability of conditioned medium (CM) from J774A.1 macrophages treated with ML-ESPs to influence the differentiation of murine myoblasts, and the mechanisms of this influence, were investigated in vitro. The results showed that the expression of Myogenic Regulatory Factors (MRFs) MyoD and myogenin, myosin heavy chain (MyHC), and the p21 cyclin-dependent kinase inhibitor were reduced in CM treated cells compared to their expression in the control group. These findings indicated that CM inhibited myoblast differentiation. Conversely, CM promoted myoblast proliferation and increased cyclin D1 levels. Taken together, results of our study suggested that CM can indirectly influence myoblast differentiation and proliferation, which provides a new method for the elucidation of the complex mechanisms involved in cell-parasite and cell–cell interactions during T. spiralis infection.

Major role of adipocyte prostaglandin E2 in lipolysis-induced macrophage recruitment

Obesity induces accumulation of adipose tissue macrophages (ATMs), which contribute to both local and systemic inflammation and modulate insulin sensitivity. Adipocyte lipolysis during fasting and weight loss also leads to ATM accumulation, but without proinflammatory activation suggesting distinct mechanisms of ATM recruitment. We examined the possibility that specific lipid mediators with anti-inflammatory properties are released from adipocytes undergoing lipolysis to induce macrophage migration. In the present study, we showed that conditioned medium (CM) from adipocytes treated with forskolin to stimulate lipolysis can induce migration of RAW 264.7 macrophages. In addition to FFAs, lipolytic stimulation increased release of prostaglandin E2(PGE2) and prostaglandin D2(PGD2), reflecting cytosolic phospholipase A2α activation and enhanced cyclooxygenase (COX) 2 expression. Reconstituted medium with the anti-inflammatory PGE2potently induced macrophage migration while different FFAs and PGD2had modest effects. The ability of CM to induce macrophage migration was abolished by treating adipocytes with the COX2 inhibitor sc236 or by treating macrophages with the prostaglandin E receptor 4 antagonist AH23848. In fasted mice, macrophage accumulation in adipose tissue coincided with increases of PGE2levels and COX1 expression. Collectively, our data show that adipocyte-originated PGE2with inflammation suppressive properties plays a significant role in mediating ATM accumulation during lipolysis.

Abstract A46: The role of circulating fibroblast precursors in promoting metastatic sarcoma

Abstract Soft tissue sarcomas are malignant tumors that develop in connective tissue. Treatment with standard chemotherapeutic agents is often minimally effective and approximately half of all patients with soft tissue sarcoma experience disease recurrence and die of metastatic cancer. Therefore, the purpose of this study is to gain a better understanding of the soft tissue sarcoma metastatic environment in order to lead to the development of more effective therapies. Studies have shown that the tumor microenvironment plays a pivotal role in promoting tumor metastasis and one of the most prominent cell types of the tumor microenvironment is the carcinoma associated fibroblast (CAF). Our studies, based upon a murine single hematopoietic stem cell transplantation model have demonstrated that hematopoietic stem cells (HSCs) are a novel source for CAFs and their circulating fibroblast precursors (CFPs). Our research has also shown that murine CFPs are present in the peripheral blood, increase with tumor burden, and contribute to tumor growth. In vitro studies have shown that HSC-derived CFPs enhance tumor cell proliferation, migration, and invasion, processes critical to tumor metastasis in vivo. The role of human CFPs in metastasis of soft tissue sarcoma, however, has yet to be determined. It is our hypothesis that HSC derived CFPs promote sarcoma cell proliferation, migration, and invasion. In order to determine the role of CFPs in sarcoma metastasis, we established a method for culturing fibroblasts from peripheral blood obtained from patients diagnosed with metastatic sarcoma. These cultured cells were then characterized by immunohistochemistry to profile fibroblast and hematopoietic markers. In order to address the functional role of CFPs during sarcoma metastasis, we examined the ability of conditioned media from cultured CFPs from patients with metastatic sarcoma to influence the proliferation, migration, and invasion of HT-1080 cells, a human sarcoma line. Our immunohistochemical findings demonstrate that fibroblasts can be isolated from the circulation of patients with metastatic sarcoma and results from our functional assays indicate that cultured CFPs from patients with metastatic sarcoma promote the proliferation, migration, and invasion of HT-1080 cells. Given that proliferation, migration, and invasion of tumor cells are essential steps in the metastatic cascade, these findings suggest that CFPs promote tumor progression. Ongoing studies involve analysis of CFPs at various time points based upon chemotherapeutic treatment in order to evaluate differences in numbers of CFPs, marker expression, growth factor production, and ability to promote metastasis. Long term, these studies may identify novel biomarkers for disease progression as well as treatment efficacy, potentially leading to the development of more effective therapies for soft tissue sarcomas. Citation Format: Dayvia A. Laws, Andrew S. Kraft, Lee R. Leddy, Amanda C. LaRue. The role of circulating fibroblast precursors in promoting metastatic sarcoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A46. doi:10.1158/1538-7445.CHTME14-A46

Comparison of Markers and Functional Attributes of Human Adipose-Derived Stem Cells and Dedifferentiated Adipocyte Cells from Subcutaneous Fat of an Obese Diabetic Donor

Objective: Adipose tissue is a robust source of adipose-derived stem cells (ADSCs) that may be able to provide secreted factors that promote the ability of wounded tissue to heal. However, adipocytes also have the potential to dedifferentiate in culture to cells with stem cell-like properties that may improve their behavior and functionality for certain applications. Approach: ADSCs are adult mesenchymal stem cells that are cultured from the stromal vascular fraction of adipose tissue. However, adipocytes are capable of dedifferentiating into cells with stem cell properties. In this case study, we compare ADSC and dedifferentiated fat (DFAT) cells from the same patient and fat depot for mesenchymal cell markers, embryonic stem cell markers, ability to differentiate to adipocytes and osteoblasts, senescence and telomerase levels, and ability of conditioned media (CM) to stimulate migration of human dermal fibroblasts (HDFs). Innovation and Conclusions: ADSCs and DFAT cells displayed identical levels of CD90, CD44, CD105, and were CD34- and CD45-negative. They also expressed similar levels of Oct4, BMI1, KLF4, and SALL4. DFAT cells, however, showed higher efficiency in adipogenic and osteogenic capacity. Telomerase levels of DFAT cells were double those of ADSCs, and senescence declined in DFAT cells. CM from both cell types altered the migration of fibroblasts. Despite reports of ADSCs from a number of human depots, there have been no comparisons of the ability of dedifferentiated DFAT cells from the same donor and depot to differentiate or modulate migration of HDFs. Since ADSCs were from an obese diabetic donor, reprogramming of DFAT cells may help improve a patient's cells for regenerative medicine applications.

Abstract 1632: Paracrine Hedgehog as a mediator of stromal cell steroidogenesis that contributes to progression of prostate cancer to castration resistant disease.

Abstract Androgen deprivation therapy (ADT) is frontline treatment for metastatic prostate cancer (PCa). ADT initially blocks disease progression but eventually allows the outgrowth of castration resistant disease (CRPC) that is refractory to the diminished systemic androgen levels in ADT-treated patients. CRPC is associated with acquired intratumoral steroidogenesis (IS), a process that increases the androgen levels in the local microenvironment of prostate tumors and enables further tumor growth and progression. IS has mainly been attributed to a steroidogenic process linked to CRPC cells and here we present evidence that benign tumor-associated prostate stromal cells (PrSCs) can also contribute to IS through a paracrine Hedgehog (HH) tumor microenvironment associated with increased production and release ofHedgehog ligands (Hhs) from prostate cancer cells in response to ADT (Chen, et al., Mol Cancer, 9: 89, 2010). Primary human PrSCs upregulate expression of a large repertoire of steroidogenic genes upon treatment with HH chemical agonists, SAG (100 nM) or Hg 1.5 (1 nM). These effects are Gli2-dependent (Levina, et al., Prostate, 72: 817, 2012) and they can be inhibited by the HH antagonist, KAAD-cyclopamine. Previously we showed that this upregulation significantly increased output of testosterone (T) in PrSCs fed DHEA precursor. More comprehensive analysis (via LC/MS/MS) of androgen outputs from PrSCs fed 22 OH-cholesterol showed that Hg1.5 significantly increased the outputs of Progesterone, DHEA, androsterone, T and dihydrotestosterone (DHT) showing that HH signaling activity effects outputs throughout the androgen biosynthetic pathway. Upregulation of the RDH5 gene by Hg1.5 suggests that the effects on production of DHT from PrSCs might be generated by increased synthesis through both classical and back-door pathways. On a weight-to-weight basis, Hg1.5-stimulated PrSCs produce higher T and DHT outputs than LNCaP cells or an androgen growth-independent variant, LNCaP-AI. Finally, we found that treatment of Sonic Hh-producing LNCaP cells with simvastatin reduced the ability of conditioned medium from these cells to induce steroidogenic gene expression from PrSCs. In summary, this work provides evidence for a stromal cell contribution to IS in CRPC mediated by a paracrine-active HH signaling tumor microenvironment induced by ADT-treatment and identifies potential treatment strategies to interfere with this process. Citation Format: Amy A. Lubik, Hans Adomat, Emma S. Guns, Ralph Buttyan. Paracrine Hedgehog as a mediator of stromal cell steroidogenesis that contributes to progression of prostate cancer to castration resistant disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1632. doi:10.1158/1538-7445.AM2013-1632

Activated hepatic stellate cells promote hepatocellular carcinoma development in immunocompetent mice

Activated hepatic stellate cells (HSCs) play a central role in the hepatic fibrosis and cirrhosis. Recently, HSCs were reported to have strong immune modulatory activities. However, the role of HSCs in hepatocellular carcinoma (HCC) remains unclear. In this study, we showed that HSCs could promote HCC growth both in vitro and in vivo. We examined the HSC-mediated inhibition of T-cell proliferation and the ability of conditioned medium from activated HSCs to promote the growth of murine HCC cell lines in vitro. We also assessed the immune suppression by HSCs during the development of HCC in immunocompetent mice. Cotransplantation of HSCs promoted HCC growth and progression by enhancing tumor angiogenesis and tumor cell proliferation and by creating an immunosuppressed microenvironment. Cotransplanted HSCs inhibited the lymphocyte infiltration in tumors and the spleens of mice bearing tumors, induced apoptosis of infiltrating mononuclear cells, and enhanced the expression of B7H1 and CD4(+) CD25(+) Treg cells. The immune modulation by HSCs seemed to be systemic. In conclusion, our data provide new information to support an integral role for HSCs in promoting HCC progression in part via their immune regulatory activities, and suggest that HSCs may serve as a therapeutic target in HCC.

Inhibition of uPAR and uPA reduces invasion in papillary thyroid carcinoma cells

We analyzed the expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) in papillary thyroid carcinoma (PTC) and normal thyroid tissue and examined in vitro how uPA and uPAR contribute to an invasive/metastatic phenotype, and the functional consequences of inhibiting this system. Retrospective chart review of PTC patients, followed by prospective study using previously obtained patient tissue and PTC cellular models. uPA and uPAR RNA and protein levels were analyzed in PTC patient tissue samples, PTC and normal thyroid tissue culture cells, and conditioned media (CM) using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and/or Western blotting. The plasminogen-activating ability of CM was examined using dark-quenched casein fluorimetry and casein-plasminogen gel zymography. The invasive potentials of the PTC and normal thyroid epithelial cell lines were assessed using an in vitro cellular invasion/migration system. uPA and uPAR RNA and protein levels were increased in PTC patient samples and PTC cells relative to controls. uPA and uPAR RNA were also significantly higher in patients with metastatic disease. Casein-plasminogen zymography and Western blotting demonstrated increased active uPA secreted by PTC cells compared with normal thyroid cells. Fluorimetric assays revealed that the PTC cells' CM was able to activate plasminogen, resulting in measurable casein hydrolysis. This casein hydrolysis was prevented by the addition of several specific uPA inhibitors. Finally, the in vitro invasion phenotypes of PTC cells were augmented by the addition of plasminogen, and this augmentation was reversed by inhibitory anti-uPA and anti-uPAR antibodies. These data provide new functional evidence of the uPA/uPAR system's role in PTC invasion/metastasis and demonstrate the attractiveness of uPA and uPAR as molecular biomarkers and therapeutic targets.

Abstract B88: Local delivery of breast cancer chemopreventives using mesenchymal stem cells

Abstract The overall goal of this study is to evaluate the ability of bone marrow mesenchymal stem cells (MSCs) to act as vehicles for local delivery of chemopreventives in mice that develop mammary cancer. Recently, efforts to use adult-derived stem cells to deliver therapeutic agents to tumor sites have shown great promise and we currently are extending this concept to include the delivery of gene therapy in the form of chemopreventives. Our goals in this initial project were to (1) isolate and study the behavior of transplanted MSCs in mammary glands of mice and (2) develop a gene expression system which robustly produces gene therapeutics. For gene therapy we aimed to target epidermal growth factor (EGFR/ErbB1/HER1) because activation of this growth factor pathway is a frequent component driving breast cancer proliferation and survival and recent evidence demonstrated that the EGFR pathway is more active in hyperplastic enlarged lobules compared with normal terminal ductal lobular units (AJP 2007 171, 253–262), indicating that EGFR activation is a very early event that precedes cancer development and therefore inhibiting EGFR may abrogate cancer development. To carry out this goal we developed an expression system that produces the natural inhibitory ligand of EGFR, decorin. Decorin is a small leucine rich proteoglycan with known effects in extracellular matrix assembly as well as growth inhibition via reduction in levels and activity of EGFR along with other signaling molecules. Thus far we have demonstrated the ability to isolate, culture and maintain murine MSCs from the FVB/N strain in an undifferentiated state. In addition, these MSCS do not form tumors upon transplantation into mammary glands of syngeneic mice. Furthermore, we have produced secreted decorin in Chinese hamster ovarian cells and MSCs. Finally, we demonstrate the ability of conditioned media containing decorin to inhibit mammary tumor cell proliferation in vitro. Our long-term goal is to test the efficacy of decorin-engineered MSCs as a breast cancer prevention modality in mouse models. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B88.

Epigenetic reprogramming of IGF1 and leptin genes by serum deprivation in multipotential mesenchymal stromal cells.

Recent studies on the therapeutic effect of multipotential mesenchymal stem cells (MSCs) in various models of injury have shown that paracrine factors secreted by MSCs are responsible for tissue repair with very little engraftment. In this study we tested the hypothesis that MSCs under stress undergo epigenetic modifications that direct secretion of paracrine factors responsible for tissue repair. Microarray assays of MSCs that had been deprived of serum (SD-MSCs), to induce stress, demonstrated an increase in the expression of several angiogenic, prosurvival, and antiapoptotic factors, including insulin-like growth factor 1 (IGF1) and leptin. Real-time polymerase chain reaction assays demonstrated a >200-fold increase in the expression of IGF1 and leptin in SD-MSCs. Chromatin immunoprecipitation of SD-MSCs revealed histone tail modifications consistent with transcriptional activation of IGF1 and leptin promoters in a reversible manner. To identify the functional significance of the epigenetic changes in stressed MSCs, we tested the prosurvival properties of SD-MSCs and the ability of conditioned medium from SD-MSCs to enhance survival of apoptotic cancer cells. First, we showed that SD-MSCs are more resistant to oxidative damage than MSCs using alkaline comet assays. Next, we demonstrated that conditioned medium from SD-MSCs decreased staurosporin-induced cell death in the KHOS osteosarcoma cell line, and that this effect was partially reversed by immunodepletion of IGF1 or leptin from the conditioned medium. In conclusion, we demonstrate that serum deprivation induces epigenetic changes in MSCs to upregulate the expression of the proangiogenic and antiapoptotic factors IGF1 and leptin.

Artesunate inhibits angiogenesis and downregulates vascular endothelial growth factor expression in chronic myeloid leukemia K562 cells

Artesunate (ART), a semi-synthetic derivative of artemisinin extracted from the Chinese herb Artemisia annua, is a safe and effective antimalarial drug. In the present investigation, we analyzed the inhibitory effects of ART on angiogenesis and on VEGF production in chronic myeloid leukemia (CML) K562 cells in vitro and in vivo. In order to analyze the effect of ART on VEGF secretion in K562 cells, we examined the level of VEGF secreted in conditioned media (CM) by ELISA assay. The result showed that ART could decrease the VEGF level in CM of K562 cells, even at a lower concentration (2 μmol/l, P < 0.01). The inhibitory effect of in vitro angiogenesis was tested on aortic sprouting in fibrin gel. ART could effectively suppress the stimulating angiogenic ability of CM by pretreated with K562 cells for 48 h in a time-dependent manner (days 3–14). The antiangiogenic effect of ART was further evaluated in vivo in chicken chorioallantoic membrane (CAM) neovascularization model. The result indicated that the stimulating angiogenic activity was decreased in response to the K562 cells treated with ART or the CM from K562 cells pretreated with ART in a dose-dependent manner (3–12 μmol/l). Furthermore, we analyzed the level of VEGF expression by western blot and detected the form of VEGF mRNA by RT-PCR in K562 cells. The experiments showed that ART could inhibit the VEGF expression, correlated well with the level of VEGF secreted in CM. These findings suggest that ART might present potential antileukemia effect as a treatment for CML therapy, or as an adjunct to standard chemotherapeutic regimens.

Requirement of KISS1 Secretion for Multiple Organ Metastasis Suppression and Maintenance of Tumor Dormancy

The KISS1 protein suppresses metastasis of several tumor models without blocking orthotopic tumor growth, but the mechanism remains elusive. For its role in human sexual maturation, KISS1 protein is secreted and processed to kisspeptins, which bind to the G protein-coupled receptor GPR54. We tested the hypothesis that KISS1 secretion is required for metastasis suppression via GPR54. KISS1 containing an internal FLAG epitope with (KFM) or without (KFMdeltaSS) a signal sequence was transfected into C8161.9 human melanoma cells, which do not express endogenous KISS1. Whole-cell lysates and conditioned medium from C8161.9(KFM) and C8161.9(KFMdeltaSS) cells were collected and analyzed for kisspeptins by immunoprecipitation and enzyme-linked immunosorbent assay. GPR54 levels were measured using real-time reverse transcription-polymerase chain reaction. The ability of conditioned medium from C8161.9(KFM) and C8161.9(KFMdeltaSS) cells to stimulate calcium mobilization in GPR54-expressing Chinese hamster ovary cells (CHO-G) and in C8161.9 cells was evaluated. Metastasis was monitored in athymic mice (groups of 10 per experiment) that were injected with C8161.9(KFM) or C8161.9(KFMdeltaSS) cells labeled with enhanced green fluorescent protein. Survival of mice injected with C8161.9 or C8161.9(KFM) cells was analyzed by Kaplan-Meier methods. Full-length KFM and KFMdeltaSS were detected in whole-cell lysates of C8161.9(KFM) and C8161.9(KFMdeltaSS) cells, respectively, but kisspeptins were detected only in conditioned medium of C8161.9(KFM) cells. In vivo, C8161.9(KFM), but not C8161.9(KFMdeltaSS), cells were suppressed for metastasis to lung, eye, kidney, and bone, with corresponding differences in mouse survival (median > 120 versus 42 days). C8161.9(KFM) cells seeded mouse lungs but did not form macroscopic metastases. Conditioned medium from C8161.9(KFM), but not C8161.9(KFMdeltaSS), cells stimulated calcium mobilization in CHO-G cells. GPR54 expression was low in C8161.9 cells, which were not stimulated by conditioned medium from C8161.9(KFM) cells. KISS1 secretion was required for multiple organ metastasis suppression and for maintenance of disseminated cells in a dormant state. The absence of GPR54 expression in C8161.9 cells (whose metastatic spread was suppressed by KFM) suggests that metastasis suppression is not mediated through this receptor. The results imply the existence of another KISS1 receptor and/or paracrine signaling. The findings raise the possibility that soluble KISS1, kisspeptins, or mimetics could be used to maintain tumor dormancy, rendering treatment of already disseminated tumor cells (i.e., micrometastases) a legitimate target.

Characterization of porcine oviductal epithelial cells, cumulus cells and granulosa cells-conditioned media and their ability to induce acrosome reaction on frozen-thawed bovine spermatozoa

The mammalian female reproductive tract cells have been wildely used in in vitro fertilization. We studied the secretory proteins of porcine oviductal epithelial cells (POEC), and cumulus cells (CC) co-cultured with granulosa cells (GC) in conditioned media (CM), and their ability in inducing acrosome reaction (AR) on frozen-thawed bovine spermatozoa. POEC and CC + GC were cultured in M 199 medium for 48, 96 and 144 h prior to investigation of protein secretion. The results from SDS-PAGE showed secretory proteins sizes of about 17, 22 and >220 kDa in both CM from POEC and CC + GC. To test the ability of CM from POEC and CC + GC in inducing AR, the CM was frozen for 1–3 months before incubating with frozen-thawed bovine spermatozoa. Percentages of the spermatozoa with AR were determined under an inverted microscope and it was found that the tested group 1 (incubated with fresh and frozen CM from POEC for 1–3 months) were 78.44 ± 7.25, 75.78 ± 4.41, 65.22 ± 5.59 and 50.56 ± 6.25, respectively. The tested group 2 (incubated with fresh and frozen CM from CC + GC for 1–3 months) had the percentages of the spermatozoa with AR at 88.67 ± 4.03, 82.22 ± 3.46, 71.00 ± 3.16, and 58.56 ± 4.69, respectively. Statistical analysis of all group percentages indicated that they were significantly different ( P < 0.05). Transmission electron microscope studies demonstrated that there were morphological changes on the sperm heads which resulted in the leakage of acrosin and subsequent AR. We concluded that the CM from both POEC and CC + GC efficiently increased in vitro AR on frozen-thawed bovine spermatozoa. Identification and functions of these secretory proteins are currently under investigation which may lead us to a better understanding of other beneficial effects in the utilization of both CM conditions.