Addition Of Conditioned Medium Research Articles

High glucose-induced downregulation of PTEN-Long is sufficient for proximal tubular cell injury in diabetic kidney disease

During the progression of diabetic kidney disease, proximal tubular epithelial cells respond to high glucose to induce hypertrophy and matrix expansion leading to renal fibrosis. Recently, a non-canonical PTEN has been shown to be translated from an upstream initiation codon CUG (leucine) to produce a longer protein called PTEN-Long (PTEN-L). Interestingly, the extended sequence present in PTEN-L contains cell secretion/penetration signal. Role of this non-canonical PTEN-L in diabetic renal tubular injury is not known. We show that high glucose decreases expression of PTEN-L. As a mechanism of its function, we find that reduced PTEN-L activates Akt-2, which phosphorylates and inactivate tuberin and PRAS40, resulting in activation of mTORC1 in tubular cells. Antibacterial agent acriflavine and antiviral agent ATA regulate translation from CUG codon. Acriflavine and ATA, respectively, decreased and increased expression of PTEN-L to altering Akt-2 and mTORC1 activation in the absence of change in expression of canonical PTEN. Consequently, acriflavine and ATA modulated high glucose-induced tubular cell hypertrophy and lamininγ1 expression. Importantly, expression of PTEN-L inhibited high glucose-stimulated Akt/mTORC1 activity to abrogate these processes. Since PTEN-L contains secretion/penetration signals, addition of conditioned medium containing PTEN-L blocked Akt-2/mTORC1 activity. Notably, in renal cortex of diabetic mice, we found reduced PTEN-L concomitant with Akt-2/mTORC1 activation, leading to renal hypertrophy and lamininγ1 expression. These results present first evidence for involvement of PTEN-L in diabetic kidney disease.

ULCERATIVE COLITIS-DERIVED INTESTINAL EPITHELIAL CELLS ARE PROTECTED FROM CELL DEATH BY THE IMMUNE MEDIATORS RELEASED FROM RESTING AND ACTIVATED SYSTEMIC T CELLS

Abstract Immune dysregulation is a major driver in the pathogenesis of ulcerative colitis (UC), but the specific mechanisms of disease initiated by the adaptive immune system remain unknown. While many studies have focused on the contribution of individual T cell subsets, disrupted communication between the epithelium and T cells in the underlying lamina propria is not fully understood in UC. This study examines the impact of T cells isolated from healthy participants on primary cultures of epithelial cells derived from UC donors. Blood derived human CD4+ and CD8+ T cells were isolated by immunomagnetic negative selection and activated by anti-CD3/CD28 cross-linking. Activated and resting T cells were then co-cultured with UC-derived colonic epithelial spheroids, consisting predominantly of stem cells, suspended in Matrigel. Spheroid damage and epithelial cell death were measured by histological scoring of brightfield microscopic images and zombie violet positive staining, detected using flow cytometry, respectively. Our earlier studies showed that activated CD4+ T cells co-cultured with spheroids from healthy donors stimulated significant spheroid damage and epithelial cell death in a dose- and time-dependent manner. In addition, we reported that the addition of conditioned media (CM) from activated CD4+ T cells to healthy spheroids also induces spheroid damage and epithelial cell death. Neither resting CD4+ nor CD8+ T cells nor activated CD8+ T cells stimulate the same response (Panel A). Our preliminary results indicate that UC-derived epithelial cells, isolated from spheroids, exhibit a higher base line of spontaneous lethal sectoring (42%), when compared to spheroids isolated from healthy colons (10%). In stark contrast to healthy spheroids, addition of CM from resting and activated CD4+ or CD8+ T cells protect the ulcerative colitis-derived spheroids from damage and isolated UC epithelial cells from spontaneous death. We have not yet identified which T cell subset are protecting UC-derived epithelial cells from damage and death. Thus, our results distinguish the healthy epithelial spheroid response to T cell CM from UC spheroids in three distinct ways: [1] CM is protective in UC, [2] Both CD4 and CD8 cells mediate this response in UC, and [3] CM from resting and activated T cells secrete the responsible immune mediators. Overall, these results signify that the biological response of UC-derived epithelial cells to immune mediators is different from that seen with healthy epithelial cells, indicating a metabolic and genetic difference in the epithelium from the UC patient.

Mechanism of synergistic inhibitory effect of benzyl isothiocyanate and zoledronic acid combination on breast cancer induction of osteoclast differentiation.

Bone is the most favored site for metastasis for each major subtype of breast cancer. Therapeutic modalities for alleviation of clinical symptoms associated with bone metastasis include surgical resection, radiation, and bone-targeted therapies, including bisphosphonates (e.g., zoledronic acid; ZA) and a humanized antibody against receptor activator of nuclear factor-κB ligand (denosumab). However, the bone-targeted therapies are expensive, and have poor pharmacokinetic attributes and/or serious adverse effects. Therefore, novel strategies are needed for treatment of bone metastasis or to increase effectiveness of existing bone-targeted therapies. We have shown previously that benzyl isothiocyanate (BITC) is a novel inhibitor of osteoclast differentiation in vitro and bone metastasis in vivo. The present study shows that BITC + ZA combination synergistically inhibits osteoclast differentiation induced by addition of conditioned media from breast cancer cells. These effects were associated with a significant increase in levels of several antiosteoclastogenic cytokines, including interferons, interleukin (IL)-3, IL-4, and IL-27. Kyoto Encyclopedia of Genes and Genomes pathway analysis of RNA-seq data from BITC and/or ZA-treated cells revealed downregulation of genes of many pathways (e.g., actin cytoskeleton, Hippo signaling, etc.) by treatment with BITC + ZA combination, but not by BITC alone or ZA alone. Confocal microscopy confirmed severe disruption of actin cytoskeleton upon treatment of MCF-7 and MDA-MB-231 cells with the BITC + ZA combination. This combination also decreased the nuclear level of yes-associated protein, a core component of Hippo signaling. In conclusion, the present study offers a novel combination for prevention or treatment of bone metastasis of breast cancer.

Abstract 3993: ERO1α as a potential target in EGFR driven non-small cell lung cancer

Abstract Endoplasmic Reticulum Oxidoreductase-1 alpha (ERO1α) is an endoplasmic reticulum (ER) resident protein responsible for oxidation of Protein Disulfide Isomerase (PDI). The molecular interactions between ERO1α and PDI allow for proper protein folding to occur. Recent reports indicate that ERO1α expression is a poor prognostic indicator of survival in multiple cancer types including multiple myeloma, lung cancer, esophageal carcinoma, hepatocellular carcinoma, and diffuse large B-cell lymphoma. In this study we utilized genetic strategies to determine the role of ERO1α in EGFR driven Non-Small Cell Lung Cancer (NSCLC). Our data indicate that CRISPR knockout of ERO1α leads to decreased clonogenicity in both PC-9 and HCC4006 cell lines. The phenotype can be partially rescued upon addition of conditioned media derived from the control cell lines. The data suggest that ERO1α expression is required for correct protein folding of a secreted factor that contributes to clonogenicity and growth. PC-9 ERO1α knockout cells injected into SCID-Beige animals led to increased overall survival (OS) compared to animals injected with PC-9 control cells. (PC-9 Control OS = 100 days, PC-9 ERO1α Knockout OS =154 days p=0.0045 **). Interestingly, ERO1α knockout cell lines still maintained sensitivity to standard of care agent Osimertinib. We identified T151742 as an ERO1α inhibitor that was approximately 2-fold more potent compared to the previously published ERO1α inhibitor EN460. A cellular thermal shift assay was performed to determine specificity of T151742 toward ERO1α compared to other FAD containing enzymes. We show that T151742 had a higher specificity towards ERO1α in comparison to other flavoenzymes such as ERO1β and LSD-1. Finally, our data indicate that PC-9 ERO1α knockout cell lines were ~2-fold resistant to T151742 by MTT but were completely resistant when using a 3D cell culture model suggesting T151742 is reliant on ERO1α in a 3D system. Finally, treatment of PC9 and HCC4006 cells with T151742 in combination with Osimertinib was synergistic in vitro. Taken together, this suggests ERO1α may be a viable target for EGFR driven NSCLC and that future studies will elucidate the use of T157142 in vivo as a single agent and in combination with EGFR inhibitors using in vivo studies. Citation Format: Brennan D. Johnson, Wei-Chih Chen, Werner J. Geldenhuys, Lori A. Hazlehurst. ERO1α as a potential target in EGFR driven non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3993.

Abstract PO-121: Investigating the role of human cancer-associated fibroblasts in pancreatic cancer invasion using patient-derived PDAC organoids

Abstract Organoid cultures have emerged as a promising research model as they more accurately recapitulate in vivo tumor features and provide a system to study cancer invasion, among others. We have previously reported the development of a human pancreatic cancer organoid model using surgically resected PDAC tumors. This model has allowed us to characterize molecular alterations critical for invasion. Culturing PDAC organoids in collagen I gels identified the ability of organoids to invade using two distinct, morphologically defined invasive phenotypes. Interestingly, invasion of PDAC organoids in collagen I decreased after culturing and passaging them in Matrigel, suggesting the microenvironment plays a crucial role in promoting invasion. To identify factors that increase PDAC organoid invasion, we sought to investigate the relationship between human PDAC organoids and patient-derived cancer-associated fibroblasts (CAFs). CAFs are a key component of the PDAC microenvironment, characterized by their ability to perform a variety of functions, including deposition and remodeling of ECM as well as promoting tumor growth, among others. CAFs may induce tumor-suppressive or tumor-promoting effects, and the role of CAFs subtypes in PDAC invasion has not been studied extensively. CAFs are also a heterogeneous group of cells: two mutually exclusive and reversible subtypes of CAFs have been discovered (inflammatory and myofibroblastic, also known as iCAFs and myCAFs respectively) driven on paracrine and juxtacrine signaling mechanisms. We have investigated the impact of patient-derived CAFs on PDAC organoid invasion by using conditioned media of CAFs in monolayers, which expand as myCAFs. After 2-3 days of culture, media was collected and human PDAC organoids (100-150 total) from 5 patients were allowed to grow and invade in collagen I gels with or without the addition of CAF conditioned media. Our results show conditioned media of CAFs increases the invasiveness of PDAC organoids but does not increase the percentage of organoids invading. In order to identify potential heterogeneity between primary CAF cultures from different patients, we performed time-lapse analyses of organoids in collagen I gels with conditioned media from 3-4 patient-derived CAFs. Interestingly, we did not find any significant differences between primary CAF cultures in inducing PDAC organoid invasion, suggesting that the ability of CAFs to induce tumor cell invasion does not vary widely between different patients. However, we did observe some patient-derived organoids did not increase invasiveness upon addition of conditioned media from any of the CAF cultures, showing heterogeneity in tumor cells from different patients in response to invasion-inducing factors secreted by CAFs. Future studies will examine the ability of iCAFs to induce invasion in our organoid model and the differences between the secretomes of myCAFs and iCAFs. Citation Format: Bernat Navarro-Serer, Kenna Sherman, Laura D. Wood. Investigating the role of human cancer-associated fibroblasts in pancreatic cancer invasion using patient-derived PDAC organoids [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-121.

Abstract PO027: Glioma cells within high functional connectivity network hubs exhibit a proliferative and infiltrative tumor phenotype

Abstract BACKGROUND: There is emerging preclinical evidence that neuronal activity influences glioma proliferation. In humans however it remains unknown how dynamic physiological interactions between glioma cells and neurons influence tumor growth and cognition. We therefore studied the mechanisms underpinning why some glioma cells maintain functional connections with neurons and the causal relationship of these connections to tumor growth and associated cognitive impairments. METHODS: We employed magnetoencephalography in newly diagnosed IDH-wild-type glioblastoma (GBM) patients to identify intratumoral regions with high (HFC) and low (LFC) brain functional connectivity. Site-directed patient tumor biopsies from HFC and LFC regions were used to perform immunohistochemistry experiments and generate patient-derived xenografts. Primary patient-derived neurosphere cultures were generated from HFC and LFC regions and spheroid invasion and EdU proliferation assays were performed to characterize their invasive and proliferative phenotypes, respectively. The morphology and size of HFC and LFC neurospheres was also evaluated to determine the clonogenicity profile. Secreted proteins from patient serum was measured using ELISA. Cognitive assessments and survival statistics of patients were performed to correlate with functional connectivity measures. RESULTS: Compared to LFC, primary patient samples from HFC regions showed increased expression of pre- and post-synaptic markers. Primary patient HFC cultures co-cultured with hippocampal neurons and induced neuron organoids again demonstrated increased synaptogenesis markers. IDH-wt GBM HFC cultures demonstrated little clonogenicitiy and low proliferation however when co-cultured with neurons markedly increased their proliferative potential both in vitro and in vivo. Similarly, when compared to HFC alone and LFC conditions, the degree of cell invasion, measured by the spheroid invasion area and microtube length, was significantly increased by the addition of conditioned media from hippocampal neuronal culture. HFC IDH-wt patient derived xenografts survived a shorter time compared to LFC. Importantly, compared to patients without intratumoral high functional connectivity regions, GBM patients with intratumoral HFC showed shorter overall survival and worse cognitive task performance. CONCLUSION: Synaptogenic glioma cells are enriched within high connectivity intratumoral regions maintains their affinity for neurons primarily through TSP-1 mediated paracrine signaling. This distinct HFC tumor sub-population exhibits an aggressive invasive and proliferative tumor phenotype, which in turn contributes to the observed adverse cognitive and survival outcomes. Citation Format: Saritha Krishna, Abrar Choudhury, Michelle Monje, Shawn Hervey-Jumper. Glioma cells within high functional connectivity network hubs exhibit a proliferative and infiltrative tumor phenotype [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO027.

Protein Digests and Pure Peptides from Chia Seed Prevented Adipogenesis and Inflammation by Inhibiting PPARγ and NF-κB Pathways in 3T3L-1 Adipocytes.

The objective was to evaluate the mechanisms of digested total proteins (DTP), albumin, glutelin, and pure peptides from chia seed (Salvia hispanica L.) to prevent adipogenesis and its associated inflammation in 3T3-L1 adipocytes. Preadipocytes (3T3-L1) were treated during differentiation with either DTP or digested albumin or glutelin (1 mg/mL) or pure peptides NSPGPHDVALDQ and RMVLPEYELLYE (100 µM). Differentiated adipocytes also received DTP, digested albumin or glutelin (1 mg/mL), before (prevention) or after (inhibition) induced inflammation by addition of conditioned medium (CM) from inflamed macrophages. All treatments prevented adipogenesis, reducing more than 50% the expression of PPARγ and to a lesser extent lipoprotein lipase (LPL), fatty acid synthase (FAS), sterol regulatory element-binding protein 1 (SREBP1), lipase activity and triglycerides. Inflammation induced by CM was reduced mainly during prevention, while DTP decreased expression of NF-κB (−48.4%), inducible nitric oxide synthase (iNOS) (−46.2%) and COX-2 (−64.5%), p < 0.05. Secretions of nitric oxide, PGE2 and TNFα were reduced by all treatments, p < 0.05. DTP reduced expressions of iNOS (−52.1%) and COX-2 (−66.4%). Furthermore, digested samples and pure peptides prevented adipogenesis by modulating PPARγ and additionally, preventing and even inhibiting inflammation in adipocytes by inhibition of PPARγ and NF-κB expression. These results highlight the effectiveness of digested total proteins and peptides from chia seed against adipogenesis complications in vitro.

Regorafenib sensitizes human breast cancer cells to radiation by inhibiting multiple kinases and inducing DNA damage

Purpose Triple-negative breast cancer (TNBC) is the most challenging and aggressive subtype of breast cancer with limited treatment options because of tumor heterogeneity, lack of druggable targets and therapy resistance. TNBCs are characterized by overexpression of growth factor receptors such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and platelet derived growth factor receptor (PDGFR) making them promising therapeutic targets. Regorafenib is an FDA approved oral multi-kinase inhibitor that blocks the activity of multiple protein kinases including those involved in the regulation of tumor angiogenesis [VEGFR1-3, TIE2], tumor microenvironment [PDGFR-β, FGFR] and oncogenesis (KIT, RET, RAF-1, BRAF). In the current study, we examined the radiosensitizing effects of Regorafenib on TNBC cell lines and explored the mechanism by which Regorafenib enhances radiosensitivity. Methods MDA-MB-231 and SUM159PT (human TNBC cell lines) and MCF 10a (human mammary epithelial cell line) were treated with Regorafenib, ionizing radiation or a combination of both. Following treatment with Regorafenib and radiation we conducted clonogenic assay to determine radiosensitivity, immunoblot analysis to assess the effect on key signaling targets, tube formation to evaluate effect on angiogenesis and comet assay as well as western blot for γH2AX to assess DNA damage response (DDR). Results Regorafenib reduced cell proliferation and enhanced radiosensitivity of MDA-MB-231 and SUM159PT cell lines but had no effect on the MCF 10a cells. Clonogenic survival assays showed that the surviving fraction at 2 Gy for both MDA-MB-231 and SUM159PT was reduced from 66.4 ± 8.9 and 88.2 ± 1.7 in controls to 38.1 ± 4.9 and 75.1 ± 1.1 following a 24 hr pretreatment with 10 μM and 5 μM Regorafenib, respectively. A marked reduction in the expression of VEGFR, PDGFR, EGFR and the downstream target, ERK, was observed with Regorafenib treatment alone or in combination with radiation. We also observed a significant inhibition of VEGF-A production in the TNBC cell lines following treatment with Regorafenib. Further, the addition of conditioned medium from Regorafenib-treated tumor cells onto human umbilical vein endothelial cells (HUVEC) suppressed tube formation, indicating an inhibition of tumor angiogenesis. Regorafenib also decreased migration of TNBC cells and suppressed radiation-induced DNA damage repair in a time-dependent manner. Conclusions Our findings demonstrate that Regorafenib enhanced radiosensitivity of breast cancer cells by inhibiting the expression of multiple receptor tyrosine kinases, VEGF-mediated angiogenesis and DNA damage response in TNBC. Therefore, combining Regorafenib with radiation and antiangiogenic agents will be beneficial and effective in controlling TNBC.

A Novel Sulforaphane-Regulated Gene Network in Suppression of Breast Cancer-Induced Osteolytic Bone Resorption.

Bone is the most preferred site for colonization of metastatic breast cancer cells for each subtype of the disease. The standard of therapeutic care for breast cancer patients with bone metastasis includes bisphosphonates (e.g., zoledronic acid), which have poor oral bioavailability, and a humanized antibody (denosumab). However, these therapies are palliative, and a subset of patients still develop new bone lesions and/or experience serious adverse effects. Therefore, a safe and orally bioavailable intervention for therapy of osteolytic bone resorption is still a clinically unmet need. This study demonstrates suppression of breast cancer-induced bone resorption by a small molecule (sulforaphane, SFN) that is safe clinically and orally bioavailable. In vitro osteoclast differentiation was inhibited in a dose-dependent manner upon addition of conditioned media from SFN-treated breast cancer cells representative of different subtypes. Targeted microarrays coupled with interrogation of The Cancer Genome Atlas data set revealed a novel SFN-regulated gene signature involving cross-regulation of runt-related transcription factor 2 (RUNX2) and nuclear factor-κB and their downstream effectors. Both RUNX2 and p65/p50 expression were higher in human breast cancer tissues compared with normal mammary tissues. RUNX2 was recruited at the promotor of NFKB1 Inhibition of osteoclast differentiation by SFN was augmented by doxycycline-inducible stable knockdown of RUNX2. Oral SFN administration significantly increased the percentage of bone volume/total volume of affected bones in the intracardiac MDA-MB-231-Luc model indicating in vivo suppression of osteolytic bone resorption by SFN. These results indicate that SFN is a novel inhibitor of breast cancer-induced osteolytic bone resorption in vitro and in vivo.

Comparative impact analysis of neuronal and glial progenitors conditioned medium on cerebellar neurons under glutamate exitotoxicity

One of the main causes of cell death in neurodegenerative diseases is excitotoxicity. Today the potential directions of treatment neurodegenerative diseases are including cell therapy, the purpose of which is to replace lost nerve tissue with donor cells. Transplanted cells along with replaced lost tissues have a paracrine effect, which requires careful study. The aim of this work was to study the effect of conditioned media, obtaining from neuronal and glial progenitor cells, on a primary culture of cerebellar neurons in a model of glutamate excitotoxicity. The cell viability, expression of marker genes for apoptosis and neuritogenesis, and the number of necrotic and apoptotic cells were determined in the culture of cerebellar neurons. The composition of the studied conditioned media was analyzed for the content of neurotrophins. A comparative analysis was revealed differences in the secretion of neurotrophins between the obtained cultures: the amount of brain-derived neurotrophic factor, nerve growth factor, ciliary neurotrophic factor and glial neurotrophic factor was higher in the secretion of glial progenitors. It was shown that the addition of conditioned media from neuronal cells does not significantly affect the viability of cerebellar neurons, whereas preincubation with media from glial progenitors has a neuroprotective effect by increasing the viability of cerebellar neurons, and during long-term cultivation promotes the growth of neurites by increasing the expression level of MAP2 and GAP43 genes.

DPF is a cell-density sensing factor, with cell-autonomous and non-autonomous functions during Dictyostelium growth and development

BackgroundCellular functions can be regulated by cell-cell interactions that are influenced by extra-cellular, density-dependent signaling factors. Dictyostelium grow as individual cells in nutrient-rich sources, but, as nutrients become depleted, they initiate a multi-cell developmental program that is dependent upon a cell-density threshold. We hypothesized that novel secreted proteins may serve as density-sensing factors to promote multi-cell developmental fate decisions at a specific cell-density threshold, and use Dictyostelium in the identification of such a factor.ResultsWe show that multi-cell developmental aggregation in Dictyostelium is lost upon minimal (2-fold) reduction in local cell density. Remarkably, developmental aggregation response at non-permissive cell densities is rescued by addition of conditioned media from high-density, developmentally competent cells. Using rescued aggregation of low-density cells as an assay, we purified a single, 150-kDa extra-cellular protein with density aggregation activity. MS/MS peptide sequence analysis identified the gene sequence, and cells that overexpress the full-length protein accumulate higher levels of a development promoting factor (DPF) activity than parental cells, allowing cells to aggregate at lower cell densities; cells deficient for this DPF gene lack density-dependent developmental aggregation activity and require higher cell density for cell aggregation compared to WT. Density aggregation activity co-purifies with tagged versions of DPF and tag-affinity-purified DPF possesses density aggregation activity. In mixed development with WT, cells that overexpress DPF preferentially localize at centers for multi-cell aggregation and define cell-fate choice during cytodifferentiation. Finally, we show that DPF is synthesized as a larger precursor, single-pass transmembrane protein, with the p150 fragment released by proteolytic cleavage and ectodomain shedding. The TM/cytoplasmic domain of DPF possesses cell-autonomous activity for cell-substratum adhesion and for cellular growth.ConclusionsWe have purified a novel secreted protein, DPF, that acts as a density-sensing factor for development and functions to define local collective thresholds for Dictyostelium development and to facilitate cell-cell communication and multi-cell formation. Regions of high DPF expression are enriched at centers for cell-cell signal-response, multi-cell formation, and cell-fate determination. Additionally, DPF has separate cell-autonomous functions for regulation of cellular adhesion and growth.

Substance P improves MSC-mediated RPE regeneration by modulating PDGF-BB

Stem cells have regenerative potentials that can be used for the treatment of critical and incurable diseases. Age-related macular degeneration (ARMD) and diabetic retinopathy are one of the most severe retinal disorders, which are mostly attributed to impairment of retinal pigmented epithelium (RPE). Thus, restoration of RPE is the main therapeutic approach to prevent the development of ocular diseases, such as ARMD. In this study, we have investigated the role of substance P (SP) on bone marrow mesenchymal stem cell (MSC)-mediated RPE regeneration in vitro. The MSCs were primed with SP followed by the addition of conditioned medium (MSCSP−CM) to RPE. The effects of MSCSP−CM on RPE activity was evaluated by assessing viability, proliferation rate, and migration of RPE. Ex vivo long-term culture led to altered cellular characteristics of MSCs by weakening cell viability, cytokine secretion, and differentiation potential. The conditioned medium of early passage MSC (E-MSCCM) enhanced the RPE viability and migration, whereas the late passage MSC (L-MSCCM) hardly influenced the RPE activity. SP priming, however, facilitated the inductive effects of MSC, and SP effect was more distinct in the late passage than in the early passage. Moreover, it was revealed that SP could exert its effects by modulating PDGF-BB secretion in the MSCs.Taken together, these results suggested that SP could restore the therapeutic effects of MSCs on retinal diseases by elevating their proliferative and paracrine activities through PDGF-PDGFR signaling in ex vivo culture.

Substance P improves MSC-mediated RPE regeneration by modulating PDGF-BB

Background & Aim Stem cells have regenerative potentials that can be used for the treatment of critical and incurable diseases. Age-related macular degeneration (ARMD) and diabetic retinopathy are one of the most severe retinal disorders, which are mostly attributed to impairment of retinal pigmented epithelium (RPE). Thus, restoration of RPE is the main therapeutic approach to prevent the development of ocular diseases, such as ARMD. Methods, Results & Conclusion In this study, we have investigated the role of substance P (SP) on bone marrow mesenchymal stem cell (MSC)-mediated RPE regeneration in vitro. The MSCs were primed with SP followed by the addition of conditioned medium (MSCSP-CM) to RPE. The effects of MSCSP-CM on RPE activity was evaluated by assessing viability, proliferation rate, and migration of RPE. Ex vivo long-term culture led to altered cellular characteristics of MSCs by weakening cell viability, cytokine secretion, and differentiation potential. The conditioned medium of early passage MSC (E-MSCCM) enhanced the RPE viability and migration, whereas the late passage MSC (L-MSCCM) hardly influenced the RPE activity. SP priming, however, facilitated the inductive effects of MSC, and SP effect was more distinct in the late passage than in the early passage. Moreover, it was revealed that SP could exert its effects by modulating PDGF-BB secretion in the MSCs. Taken together, these results suggested that SP could restore the therapeutic effects of MSCs on retinal diseases by elevating their proliferative and paracrine activities through PDGF-PDGFR signaling in ex vivo culture.

Effects of Neurotrophic Factors in Glial Cells in the Central Nervous System: Expression and Properties in Neurodegeneration and Injury.

Astrocytes, oligodendrocytes, and microglia are abundant cell types found in the central nervous system and have been shown to play crucial roles in regulating both normal and disease states. An increasing amount of evidence points to the critical importance of glia in mediating neurodegeneration in Alzheimer’s and Parkinson’s diseases (AD, PD), and in ischemic stroke, where microglia are involved in initial tissue clearance, and astrocytes in the subsequent formation of a glial scar. The importance of these cells for neuronal survival has previously been studied in co-culture experiments and the search for neurotrophic factors (NTFs) initiated after finding that the addition of conditioned media from astrocyte cultures could support the survival of primary neurons in vitro. This led to the discovery of the potent dopamine neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF). In this review, we focus on the relationship between glia and NTFs including neurotrophins, GDNF-family ligands, CNTF family, and CDNF/MANF-family proteins. We describe their expression in astrocytes, oligodendrocytes and their precursors (NG2-positive cells, OPCs), and microglia during development and in the adult brain. Furthermore, we review existing data on the glial phenotypes of NTF knockout mice and follow NTF expression patterns and their effects on glia in disease models such as AD, PD, stroke, and retinal degeneration.

Pharmacological Inhibition of NF\u03baB Reduces Prostate Cancer Related Osteoclastogenesis In Vitro and Osteolysis Ex Vivo

NFκB is implicated in cancer and bone remodelling, and we have recently reported that the verified NFκB inhibitor Parthenolide (PTN) reduced osteolysis and skeletal tumour growth in models of metastatic breast cancer. Here, we took advantage of in vitro and ex vivo bone cell and organ cultures to study the effects of PTN on the ability of prostate cancer cells and their derived factors to regulate bone cell activity and osteolysis. PTN inhibited the in vitro growth of a panel of human, mouse and rat prostate cancer cells in a concentration-dependent manner with a varying degree of potency. In prostate cancer cell—osteoclast co-cultures, the rat Mat-Ly-Lu, but not human PC3 or mouse RM1-BT, enhanced RANKL stimulated osteoclast formation and PTN reduced these effects without affecting prostate cancer cell viability. In the absence of cancer cells, PTN reduced the support of Mat-Ly-Lu conditioned medium for the adhesion and spreading of osteoclast precursors, and survival of mature osteoclasts. Pre-exposure of osteoblasts to PTN prior to the addition of conditioned medium from Mat-Ly-Lu cells suppressed their ability to support the formation of osteoclasts by inhibition of RANKL/OPG ratio. PTN enhanced the ability of Mat-Ly-Lu derived factors to increase calvarial osteoblast differentiation and growth. Ex vivo, PTN enhanced bone volume in calvaria organ—Mat-Ly-Lu cell co-culture, without affecting Mat-Ly-Lu viability or apoptosis. Mechanistic studies in osteoclasts and osteoblasts confirmed that PTN inhibit NFκB activation related to derived factors from Mat-Ly-Lu cells. Collectively, these findings suggest that pharmacological inhibition of the skeletal NFκB signalling pathway reduces prostate cancer related osteolysis, but further studies in the therapeutic implications of NFκB inhibition in cells of the osteoblastic lineage are needed.

MiRNA influences in mesenchymal stem cell commitment to neuroblast lineage development.

Mesenchymal Stem Cells (MSCs) are widely used in therapeutic applications. Their plasticity and predisposition to differentiate into a variety of cell types, including those of the neuronal lineage, makes them ideal to study whether a selection of miRNAs may direct the differentiation of MSCs into neuroblasts or neuroblastoma to mature neurons. Following a short-listing, miR-107, 124 and 381 were selected as the most promising candidates for this differentiation. MSCs differentiated into cells of the neural lineage (Conditioned Cells) upon addition of conditioned medium (rich in microvesicles containing miRNAs) obtained from cultured SH-SY5Y neuroblastoma cells. Characterisation of stemness (including SOX2, OCT4, Nanog and HCG) and neural markers (including Nestin, MASH1, TUBB3 and NeuN1) provided insight regarding the neuronal state of each cell type. This was followed by transfection of the three miRNA antagonists and mimics, and quantification of their respective target genes. MiRNA target gene expression following transfection of MSCs with miRNA inhibitors and mimics demonstrated that these three miRNAs were not sufficient to induce differentiation. In conditioned cells the marginal changes in the miRNA target expression levels reflected potential for the modulation of intermediate neural progenitors and immature neuron cell types. Transfection of various combinations of miRNA inhibitors and/or mimics revealed more promise. Undoubtedly, a mix of biomolecules is being released by the SH-SY5Y in culture that induce MSCs to differentiate. Screening for those biomolecules acting synergistically with specific miRNAs will allow further combinatorial testing to elucidate the role of miRNA modulation.

Suppressive effect of mesenchymal stromal cells on interferon-γ-producing capability of spleen cells was specifically enhanced through humoral mediator(s) from mouse oral squamous cell carcinoma Sq-1979 Cells In Vitro

Aim: The aim of this study was to compare the immunomodulatory effects in the tumor milieu of mouse oral squamous cell carcinoma (OSCC) cells harboring primary and advanced phenotypes. We established an in vitro co-culture system using mouse OSCC cells, spleen cells, and mesenchymal stromal cells. Methods: Sq-1979 is an OSCC cell line derived from C3H mice; 233-11 cells were established from a primary Sq-1979 tumor; L3–5, L5–11, and L6–8 cells were established from lymph node-metastasized Sq-1979 cells. 10T1/2 is a fibroblast line derived from C3H mice. The OSCC cells were co-cultured with anti-CD3 antibody-stimulated mouse spleen cells in the presence or absence of 10T1/2 cells, and the producing capability of interferon (IFN)-γ and interleukin (IL)-10 was evaluated using enzyme-linked immunosorbent assay. Results: The production of IFN-γ by the stimulated spleen cells was specifically enhanced in the presence of co-cultured L-cells. The production of IL-10 was conversely reduced in the co-culture with all the OSCC cell lines used. The production of IFN-γ was significantly reduced in the co-culture with directly contacted 10T1/2 cells, and further reduction was observed in the presence of Sq-1979 and 233-11 cells but remained unchanged in the presence of L-cells. The reduction of IFN-γ production was also observed by the addition of conditioned medium from the Sq-1979-1 cells. Conclusion: Sq-1979 cells specifically enhanced the immune-suppressive activity of mesenchymal stromal cells through humoral factor (s).

High glucose-induced defective thrombospondin-1 release from astrocytes via TLR9 activation contributes to the synaptic protein loss

Diabetes, characterized by chronic hyperglycemia, is known to induce synaptic degeneration in the brain, thereby resulting in cognitive dysfunction. Thrombospondin-1(TSP-1), the secreted protein produced by astrocytes, plays a crucial role in promoting synapse formation. Toll-like receptor 9 (TLR9) has been widely known to initiate the innate immune response. We recently reported TLR9 activation in neurons results in tau hyperphosphorylation induced by HG in vitro. Its activation has been also considered to mediate oxidative stress and astrocytic dysfunction under pathological circumstance. However, whether astrocytic TSP-1 alteration plays a role in synaptic protein loss under high glucose condition and whether TLR9 activation is involved in this process have not been reported. In this study, we found that primary mouse astrocytes incubated in high glucose (30mM) induced a significant decreased TSP-1 secretion and increased intracellular contents of TSP-1 without affecting transcription level. Addition of conditioned medium from high glucose (30mM) treated astrocytes to the primary neurons exhibited reduced synaptic proteins expression, which was attenuated by treatment with exogenous rTSP-1. In addition, we demonstrated that TLR9 activation along with reactive oxygen species (ROS) generation in astrocytes was induced by high glucose (30mM). Furthermore, we explored the relationship between TLR9 activation and TSP-1 production. Both TLR9 deficiency and the antioxidant N-acetyl-L-cysteine treatment improved altered intra- and extracellular TSP-1 levels under high glucose condition. Together, our findings suggest that high glucose (30mM) impairs TSP-1 secretion from astrocytes, which depends on astrocytic dysfunction associated with TLR9 activation mediated ROS signaling, ultimately contributing to the synaptic proteins loss.

Abstract 3058: Syndecan-1 mediates breast cancer metastasis to the brain through IL-8 and PECAM-1 signaling

Abstract Brain metastasis is a devastating, late-stage event affecting approximately 10-30% of breast cancer patients. However, it is not well understood how breast cancer cells migrate across the blood-brain barrier (BBB) and invade into the brain. Syndecans (Sdcs) are cell surface heparan sulfate proteoglycans (HSPGs) that have previously been linked to breast cancer progression and metastasis. Preliminary findings from our lab indicate that Sdc1 may be involved in breast cancer metastasis to the brain. In clinical samples from breast cancer patients with brain metastases, 67% of the brain metastasis samples stained positive for Sdc1 expression. In an experimental mouse model of metastasis, we found that silencing expression of Sdc1 in MDA-231 breast cancer cells greatly reduced metastasis specifically to the brain, with no difference in lung or bone metastases. Additionally, silencing expression of Sdc1 in MDA-231 cells resulted in a reduction in cancer cell migration across an in vitro BBB transwell model system. These findings prompted us to investigate the mechanism through which Sdc1 facilitates breast cancer cell migration across the BBB. Since BBB endothelial cell junctions have been reported to be disrupted in metastasis to the brain, immunofluorescence was performed to examine localization of junction proteins in our BBB model following addition of conditioned medium (CM) from MDA-231 non-silenced (NS1) control and Sdc1KD cells. We observed disruption in PECAM-1 localization at BBB endothelial cell junctions upon addition of CM from MDA-231 NS1 cells with markedly less disruption occurring upon treatment with CM from Sdc1KD cells. These results suggest that paracrine factors secreted from MDA-231 cells may facilitate breast cancer cell migration across the BBB by affecting PECAM-1 localization on BBB endothelial cells. By performing a cytokine array using CM from MDA-231 cells, we determined that Sdc1KD CM contained lower levels of IL-8 than MDA-231 NS1 cell CM. These findings were confirmed by ELISA, qRT-PCR, and multiplex analysis. We then went on to treat endothelial cells in the xCELLigence in vitro BBB model system with IL-8 and observed a sufficient decrease in cell index readings, suggesting that IL-8 affects BBB barrier permeability. Taken together, our results suggest that Sdc1 supports breast cancer cell migration across the BBB through a signaling mechanism involving IL-8 and PECAM-1. Elucidating this mechanism will allow for the development of therapeutic strategies to combat breast cancer cell metastasis to the brain. [S.M.W. and M.S. contributed equally to this work.] Citation Format: Sierra Mosticone Wangensteen, Megan Sayyad, Madhavi Puchalapalli, Megan Sullivan, Jamie Singh, Briana Ratchford, Jayda Abrams, Majid Jahromi, Bin Hu, Michael Idowu, Jennifer Koblinski. Syndecan-1 mediates breast cancer metastasis to the brain through IL-8 and PECAM-1 signaling [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 3058. doi:10.1158/1538-7445.AM2017-3058

Unique properties of thymic antigen-presenting cells promote epigenetic imprinting of alloantigen-specific regulatory T cells.

Regulatory T cells (Tregs) are potential immunotherapeutic candidates to induce transplantation tolerance. However, stability of Tregs still remains contentious and may potentially restrict their clinical use. Recent work suggested that epigenetic imprinting of Foxp3 and other Treg-specific signature genes is crucial for stabilization of immunosuppressive properties of Foxp3+ Tregs, and that these events are initiated already during early stages of thymic Treg development. However, the mechanisms governing this process remain largely unknown. Here we demonstrate that thymic antigen-presenting cells (APCs), including thymic dendritic cells (t-DCs) and medullary thymic epithelial cells (mTECs), can induce a more pronounced demethylation of Foxp3 and other Treg-specific epigenetic signature genes in developing Tregs when compared to splenic DCs (sp-DCs). Transcriptomic profiling of APCs revealed differential expression of secreted factors and costimulatory molecules, however neither addition of conditioned media nor interference with costimulatory signals affected Foxp3 induction by thymic APCs in vitro. Importantly, when tested in vivo both mTEC- and t-DC-generated alloantigen-specific Tregs displayed significantly higher efficacy in prolonging skin allograft acceptance when compared to Tregs generated by sp-DCs. Our results draw attention to unique properties of thymic APCs in initiating commitment towards stable and functional Tregs, a finding that could be highly beneficial in clinical immunotherapy.