Secretome Analysis of Rabbit and Human Mesenchymal Stem and Endothelial Progenitor Cells: A Comparative Study

Human adipose tissue-derived mesenchymal stem cells (AT-MSCs) have been studied several years for their immunomodulatory effect through the paracrine mechanism and cytokine secretion. In combination with endothelial progenitor cells (EPCs), MSCs have great therapeutical potential for the repair of endothelium and wound healing. However, little is known about the cytokine profile of rabbit AT-MSCs or even EPCs. The aim of this study was to analyze the secretomes of these rabbit stem/progenitor cells. A large-scale human cytokine array (up to 80 cytokines) was used to identify and compare cytokines secreted into conditioned media of human and rabbit AT-MSCs as well as HUVECs and rabbit EPCs. Few cytokines were highly expressed by human AT-MSCs (TIMP-2, TIMP-1), HUVECs (MCP-1, TIMP-2, GRO, Angiogenin, IL-8, TIMP-1), or by rabbit EPCs (TIMP-2). Several cytokines have moderate expression by human (MCP-1, GRO, Angiogenin, TGF-β 2, IL-8, LIF, IL-6, Osteopontin, Osteoprotegerin) and rabbit AT-MSCs (TIMP-2, TGF-β 2, LIF, Osteopontin, IL-8, IL-5, IL-3) or by HUVECs (IL-6, MIF, TGF-β 2, GCP-2, IGFBP-2, Osteoprotegerin, EGF, LIF, PDGF-BB, MCP-3, Osteopontin, Leptin, IL-5, ENA-78, TNF- β) and rabbit EPCs (TGF-β 2, Osteopontin, GRO, LIF, IL-8, IL-5, IL-3). In conclusion, the proposed method seems to be useful for the secretome analysis of rabbit stem/progenitor cells.

Circulating Cytokines Present in Multiple Myeloma Patients Inhibit the Osteoblastic Differentiation of Adipose and Bone Marrow Stem Cells

Introduction: Myeloma is characterized by bone lesions, which are related to both an increased osteoclast activity and a defect in the differentiation of medullary mesenchymal stem cells (MSCs) into osteoblasts. Outside the medullary environment, adipocyte-derived MSCs (ASCs) could represent a source of functional osteoblasts. However, we recently found a defect in the osteoblastic differentiation of ASCs from myeloma patients (MM-ASCs). We therefore examined the effects of plasma from myeloma patients at diagnosis (MM-plasmas) and in complete remission (CR-plasmas) and from healthy donors on the osteoblastic differentiation of healthy donor-derived ASCs (HD-ASCs) and healthy donor-bone marrow derived MSCs (HD-BM-MSCs). Materials and Methods: We studied 11 MM-ASCs, 5 HD-ASCs and 3 HD-BM-MSCs. The plasmas were from myeloma patients with bone lesions at diagnosis (n=12), in complete remission (n=8) and from 5 pools of 100 healthy donors (HD-plasmas). HD-ASCs were differentiated into osteoblasts and adipocytes and HD-BM-MSCs into osteoblasts with the three types of plasmas as well as newly discovered cytokines. Results: Osteoblastogenesis in HD-ASCs was suppressed by MM-plasmas. Alizarin red coloration and alkaline phosphatase activity were strongly decreased along with a decreased RUNX2 and osteocalcin expression. However, adipocyte differentiation was unaltered. The osteoblastic differentiation deficiency was reversible once the plasma-derived factors were removed. Using cytokine array and comparing MM-plasmas with HD-plasmas, we identified seven cytokines (ANG1, ENA-78, EGF, PDGF-AA/AB/BB and TARC), besides DKK1, highly increased in MM-plasmas which was confirmed by ELISA (Figure). They separately inhibited the osteoblastic differentiation of HD-ASCs. In contrast, myeloma patients in remission had a cytokine plasma level almost normal with barely no osteoblastic differentiation inhibition. In addition, the mixture of the 7 cytokines with and without DKK1 inhibited not only the HD-ASCs but also the HD-BM-MSCs. Concomittantly, we observed that MM-plasmas enhanced adipogenesis-related gene expression. Comparison of MM-ASCs and HD-ASCs by RNA sequencing showed that two master genes characterizing adipocyte differentiation, CD36 and PPARγ, were upregulated in MM-ASCs as compared to HD-ASCs. Moreover, we demonstrated a significant increase in CD36 and PPARγ expression in HD-ASCs in the presence of MM-plasmas or the seven cytokines individually, similarly as in MM-ASCs. Finally, we tried to identify the origin of these cytokines. When myeloma patients were in remission, the cytokines levels were strongly decreased suggesting a malignant plasmocyte secretion. This was reinforced by the detection of the 7 cytokines in three different myeloma cell lines with an especially high secretion of PDGF-AA. We conclude that specific cytokines in MM-plasmas, besides the well-known DKK1, inhibit the osteoblastic differentiation of MM- and HD-ASCs with a skewing towards adipocyte differentiation. Of note, this inhibition by the cytokines were also observed on HD-BM-MSCs suggesting that this could also be the case on myeloma-BM-MSCs. Legend to figure: Cytokine expression in MM, CR and HD-plasmas (A) Representative images of cytokine array blots probed with the plasma samples. The red boxes identify the cytokines significantly dysregulated in MM- as compared to HD- or CR-plasmas and further analyzed by ELISA. The blue boxes identified the cytokines similarly expressed in MM-/CR-/HD-plasmas. (B) Cytokine concentrations in the HD-plasmas (n=5), MM-plasmas (n=11) and CR-plasmas (n=8) were measured by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001, ns (not significant). Figure 1 Figure 1. Disclosures Delhommeau: Novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy. Garderet: Celgene: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Sanofi: Consultancy; Takeda: Consultancy.

Multi-proteomic Analysis Revealed Distinct Protein Profiles in Cerebrospinal Fluid of Patients Between Anti-NMDAR Encephalitis NORSE and Cryptogenic NORSE

Background: New-onset refractory status epilepticus (NORSE) caused by anti-N-methyl-d-aspartate receptor (anti-NMDAR) encephalitis and cryptogenic etiologies are various in clinical features. The underlying mechanisms of the diseases have still remained elusive.Methods: 6 patients with anti-NMDAR encephalitis NORSE, 5 with cryptogenic NORSE (C-NORSE), and 5 controls were enrolled. Clinical data, including clinical features, cerebrospinal fluid (CSF) samples, and cranial images were collected. CSF samples were tested for FAIMS based quantitative proteomic analysis, immunome protein microarray, and inflammatory cytokine array. Bioinformatics and immunostaining were used for interpretation and verification.Results: The clinical features of anti-NMDAR encephalitis NORSE and C-NORSE patients were similar to those reported previously. Proteomic analysis revealed that 101 proteins (63 up-regulated and 38 down-regulated) and 56 proteins (42 up-regulated and 14 down-regulated) changed in CSF samples of anti-NMDAR encephalitis NORSE and C-NORSE patients, respectively. The mean fold-change of the up-regulated proteins, namely up-proteomic score in this study, was positively associated with the severity of disease, including ICU stay, mRS score at discharge, and time taken for patients awaking from a coma. The distribution of changed proteins in CSF between the two diseases were quite different. Pathways of humoral immune response, wound healing, and epigenetic regulation of transcription were up-regulated in anti-NMDAR encephalitis NORSE. Pathways of innate and lymphocyte mediated immune response, synaptic functions, ubiquitination, and cell apoptosis were up-regulated in C-NORSE group, suggesting that C-NORSE could undergo neurodegeneration. This was consistent with a mouse model of SE, which showed high ubiquitination and cell apoptosis in CA1 to CA3 regions of hippocampus. Immunome microarray analysis demonstrated high autoantibody targeting 48 proteins in CSF samples of anti-NMDAR encephalitis NORSE. However, in C-NORSE, the reaction was kept at a very low level. Inflammatory cytokine array, unexpectedly, showed no remarkable changes except for decreased level of interleukin-6 (IL-6) in both anti-NMDAR encephalitis NORSE and C-NORSE.Conclusions: The present study suggests that the up-proteomic score of CSF could be a promising indicator for assessment of the severity of anti-NMDAR encephalitis NORSE and C-NORSE. The distinct CSF proteomes imply different pathogenic mechanisms of the two diseases, and immunotherapy strategies as well.

P16.12 Proangiogenic effect of fibroblast activation protein positive stromal cells derived from human glioblastomas

BACKGROUND Increased expression of fibroblast activation protein (FAP) is characteristic for several cancer types including human glioblastomas (GBM). FAP is expressed on both cancer as well as stromal cells which were demonstrated in extracranial tumors to maintain a microenvironment permissive for tumor growth and thus to contribute to tumor progression. FAP is considered a potential diagnostic and therapeutic target and several approaches for its targeting have been recently developed. In this work, we investigated the role of FAP+ stromal cells in glioblastoma angiogenesis. MATERIAL AND METHODS Expression of FAP and other phenotypic markers was assessed by immunocytochemistry and immunohistochemistry. FAP+ stromal cells and primary microvascular endothelial cells were isolated using magnetic activated cell sorting (MACS). Genetic aberrations were assayed by comparative genomic hybridization/single-nucleotide polymorphism analysis. Angiogenesis was evaluated using a 3D spheroid-based sprouting assay and a chorioallantoic membrane assay. A cytokine array was used to analyze soluble mediators released by FAP+ stromal cells. RESULTS FAP+ stromal cells were predominantly localized around activated CD105+ endothelial cells and their quantity positively correlated with glioblastoma vascularization. FAP+ stromal cells derived from human GBMs had a mesenchymal phenotype, were non-tumorigenic and in most cases lacked cytogenetic aberrations characteristic of GBMs. Conditioned media derived from FAP+ stromal cells induced angiogenic sprouting of both macrovascular HUVEC as well as microvascular primary endothelial cells derived from human GBMs. In a chorioallantoic membrane assay, admixture of FAP+ stromal cells to glioma cells was associated with increased angiogenesis and more frequent occurrence of hemorrhages. Cytokine array revealed a significant disbalance between several proangiogenic and antiangiogenic mediators compared to normal pericytes, and an increased Angiopoietin 2/1 ratio in conditioned media from FAP+ stromal cells. CONCLUSION Our results bring new evidence that GBM associated FAP+ stromal cell promote angiogenesis by changing the balance between proangiogenic and antiangiogenic mediators and thus they may contribute to glioblastoma progression. ACKNOWLEDGEMENT Supported by Progres Q28/1LFUK and grant LM2015064 of the EATRIS-CZ and the Center for Tumor Ecology (CZ.02.1.01/0.0/0.0/16_019/0000785).

Cytotrophoblast extracellular vesicles enhance decidual cell secretion of immune modulators via TNFα

ABSTRACTThe placenta releases large quantities of extracellular vesicles (EVs) that likely facilitate communication between the embryo/fetus and the mother. We isolated EVs from second trimester human cytotrophoblasts (CTBs) by differential ultracentrifugation and characterized them using transmission electron microscopy, immunoblotting and mass spectrometry. The 100,000 g pellet was enriched for vesicles with a cup-like morphology typical of exosomes. They expressed markers specific to this vesicle type, CD9 and HRS, and the trophoblast proteins placental alkaline phosphatase and HLA-G. Global profiling by mass spectrometry showed that placental EVs were enriched for proteins that function in transport and viral processes. A cytokine array revealed that the CTB 100,000 g pellet contained a significant amount of tumor necrosis factor α (TNFα). CTB EVs increased decidual stromal cell (dESF) transcription and secretion of NF-κB targets, including IL8, as measured by qRT-PCR and cytokine array. A soluble form of the TNFα receptor inhibited the ability of CTB 100,000 g EVs to increase dESF secretion of IL8. Overall, the data suggest that CTB EVs enhance decidual cell release of inflammatory cytokines, which we theorize is an important component of successful pregnancy.

Prevention of Nonalcoholic Hepatic Steatosis by Shenling Baizhu Powder: Involvement of Adiponectin-Induced Inhibition of Hepatic SREBP-1c

Background. Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease worldwide, and its incidence is increasing annually, but there is currently no specific drug for treating NAFLD. Shenling Baizhu powder (SL) is a safe herbal compound commonly used in clinical practice. Our previous research has shown that SL has the effect of preventing NAFLD, but its specific mechanism has not been determined. In this study, the potential mechanism of SL on NAFLD was explored by in vivo experiments. Methods. Wistar rats fed a choline-deficient amino acid-defined diet (CDAA) were treated with SL for 8 weeks. Then, serum samples were collected to obtain biochemical indicators; adipose tissue and liver samples were collected for pathological detection; a moorFLPI-2 blood flow imager was used to measure liver microcirculation blood flow, and a rat cytokine array was used to screen potential target proteins. The expression of liver adiponectin/SREBP-1c pathway-related proteins was determined by Western blotting. Results. SL effectively reduced the liver wet weight, as well as the levels of total cholesterol (TC) and triglyceride (TG) in the liver, and ameliorated liver injury in CDAA-fed rats. Pathological examinations showed that SL markedly reduced liver lipid droplets and improved liver lipid accumulation. In addition, the detection of liver blood flow showed that SL increased liver microcirculation in CDAA-fed rats. Through the cytokine array, a differentially expressed cytokine, namely, adiponectin, was screened in the liver. Western blotting assays showed that SL increased the expression of adiponectin and phosphoacetyl-CoA Carboxylase (p-ACC) in the liver and decreased the expression of steroid regulatory element-binding protein-1c (SREBP-1c) and fatty acid synthase (FAS). Conclusion. These results suggest that SL can increase the levels of adiponectin in the liver and serum and can inhibit the expression of SREBP-1c, thereby regulating systemic lipid metabolism and reducing liver lipid accumulation.

BMK_PANC: Search for new predictive/prognosis biomarkers (BKs) in potentially resectable pancreatic adenocarcinoma (PDAC).

e16782 Background: New BKs are urgently needed in order to improve PDAC patient’s outcome. In this sense, the main aim of this multicenter and prospective project is to find predictive and/or prognostic BKs based on many cytokines that are produced in cancer microenvironment and can nowadays be accurately analyzed. Methods: Patients diagnosed of potentially resectable PDAC with poor prognostic factors and treated with neoadjuvant treatment are being included. Serial analyses (basal, with TC, with surgery and time of relapse/or after 1y of follow-up) of serum levels of 80 cytokines are being done. Results: With a median follow-up of 11,25 months (m) (4,67-38,28) 22 patients (pt) with median of age of 62,5 y-0, most of them men (63.6%) have been included, with radical surgery performed in 14 of them (2 pts with bypass). Seven operated pt received adjuvant treatment and 12 operated pt developed progression of disease. 18 pt have received nab-paclitaxel and gemcitabine as perioperative treatment and 4 received Folfirinox. Perineural and vascular invasion were confirmed after surgery in 45,5% and 27,3% of cases, respectively. Positive nodes were found in 40,9% of them, being the median of positive and resected nodes of 1 (0-14) and 26 respectively. Surgical margins were negative (R0) in 64,3% of cases (35,7% R1). Pathological response was described as partial in 13,6%, with no changes in 31,8% and progression in 13,6%. Globally, PFS and OS were 13,50 m (CI95%: 6,44-20,55) and 17,31 m (CI 95% 8,86-25,76). Significant differences (p = 0,002) were observed in cases in which radical surgery had been performed with PFS of 13,5 m (9,34-17,66) and 3.97 m (0-8,23) for unresectable patients. Regarding the cytokine array, we observed different PFS outcome based on basal MCP3 levels (low: 16,32 m CI 95%: 5,02-27,63; high: 7,52 m CI95%: 6,23-8.81, p = 0,023), basal Oncostatin (high: 7,12 m CI95%: 3,09-11,15, low: not reached; p = 0,007) or basal Eotaxin (low: 7,12 m CI 95%: 5,75-8,50; high: not reached, p = 0,029). A cytokine score was created based on these results, maintained significant differences for PFS (16,32 m CI95%: 5,07-27,57 vs 6,53 m CI95%: 2,75-10,32, p = 0.003). Our cytokine score showed a significant role as independent prognosis factor in multivariate analysis (HR: 0,196 CI95%: 0,042-0,918; p = 0.039). Conclusions: In potentially resectable/borderline PDAC our basal cytokine score seems to be able to discriminate as independent prognosis factor between those patients that will get significant benefit from neoadjuvant treatment from those that will not

Modulating tumor immune microenvironment by the STK11/LKB1 signaling in breast cancer.

e15185 Background: Standard treatment for breast cancer patients includes surgery, chemotherapy, radiotherapy, target and endocrine therapy. Immune checkpoint inhibitors are newly developing medications. The theoretical basis of immunotherapy is the interaction between cancer cells and tumor-infiltrating immune cells. Cancer cells secrete cytokines and create a specific tumor immune microenvironment (TIME) to attract or modulate immune cells. Further, genetic mutations or copy-number variations in cancer cells contribute to immunosuppression. Liver kinase B1 (LKB1) protein ( STK11 gene) is the upstream of AMP activated Protein Kinase (AMPK)/mammalian Target of Rapamycin Complex 1 (mTORC1) signaling pathway. STK11/LKB1 is one of the possible pathways modulating TIME. Methods: Twenty-seven breast cancer patients who developed recurrence within postoperative 24 months and 22 control cancer patients without recurrence were enrolled from National Cheng Kung University Hospital in Taiwan. Targeted deep sequencing was performed to assess the mutations among individuals with breast cancer using a panel of 143 cancer-associated genes. Bioinformatics and public databases were used to predict the protein functions of the STK11 genes. Immunohistochemical staining of LKB1 protein was performed in clinical specimens. Immune-related molecules were analyzed by RNA sequencing and cytokine array after suppression of STK11. Results: Mutations of STK11 gene were detected in recurrent patients and associated with poor prognosis of patients. From immunohistochemical study, the patients with low LKB1 expression had a worse survival. We utilized CRISPER/Cas9 system to transfect sgRNA into three mouse cell lines, including M158, NF639 and PY8119. RNA sequencing was performed in parental and Stk11-sgRNA cells. Immune-related pathways were ranked in the top 10 networks. Increased mRNA expression of Csf3 (protein G-CSF), Cxcl5, and Ccl2 was detected. The results are confirmed by cytokine array. The expression of G-CSF (gene Csf3) and CXCL5 (gene Cxcl5) proteins was increased in Stk11-sgRNA cells. The results were similar with RNA sequencing. Conclusions: Our findings suggest that suppression of STK11/LKB1 is correlated with early recurrence of breast cancer patients and contributes to modulate TIME. The STK11/LKB1 and downstream AMPK/mTORC1 pathways may be potential targets for immunotherapy.

Autophagy Modulators Profoundly Alter the Astrocyte Cellular Proteome

Autophagy is a key cellular process that involves constituent degradation and recycling during cellular development and homeostasis. Autophagy also plays key roles in antimicrobial host defense and numerous pathogenic organisms have developed strategies to take advantage of and/or modulate cellular autophagy. Several pharmacologic compounds, such as BafilomycinA1, an autophagy inducer, and Rapamycin, an autophagy inhibitor, have been used to modulate autophagy, and their effects upon notable autophagy markers, such as LC3 protein lipidation and Sequestosome-1/p62 alterations are well defined. We sought to understand whether such autophagy modulators have a more global effect upon host cells and used a recently developed aptamer-based proteomic platform (SOMAscan®) to examine 1305 U-251 astrocytic cell proteins after the cells were treated with each compound. These analyses, and complementary cytokine array analyses of culture supernatants after drug treatment, revealed substantial perturbations in the U-251 astrocyte cellular proteome. Several proteins, including cathepsins, which have a role in autophagy, were differentially dysregulated by the two drugs as might be expected. Many proteins, not previously known to be involved in autophagy, were significantly dysregulated by the compounds, and several, including lactadherin and granulins, were up-regulated by both drugs. These data indicate that these two compounds, routinely used to help dissect cellular autophagy, have much more profound effects upon cellular proteins.