Basic FGF Research Articles

The impact of cytokine change after hepatitis C virus clearance by direct antiviral agents on the risk of hepatocellular carcinoma

De novo and early recurrent hepatocellular carcinoma (HCC) have been observed in clinical practice after direct antiviral agents (DAA) treatment. The study aims to investigate the change of cytokines and growth factors after hepatitis C virus (HCV) clearance by DAAs and their impact on the risk of HCC development. The chronic hepatitis C (CHC) patients with or without HCC who received DAA treatment were prospectively enrolled. The cytokines and growth factors were measured using Bio-Plex Pro™ Human Cytokine 27-plex Assay before and 12 weeks off DAA treatment. A total of 37 patients were enrolled for final analysis. There were 11 males (29.7%) and 26 females (70.3%). The mean age was 67.39±10.48 years. 11 (29.7%) patients were HCV-related HCC patients. The HCV genotype included genotype 2 in 26 patients and genotype 1b in 10 patients, and genotype 6 in 1. Among them, 35 (94.6%) patients achieved sustained virologic response (SVR). Two patients with HCC failed to DAA treatment. In HCV-related HCC patients, serum IP-10 level significantly declined after HCV clearance, but no difference in five growth factors including G-CSF, GM-CSF, basic FGF, PDGF-BB, and VEGF. Several cytokines including IP-10 significantly declined after HCV clearance in CHC patients. This study showed only serum IP-10 level, a risk factor of HCC, was significantly declined after HCV clearance and no change in the markers of growth factors in HCV-related HCC patients, suggesting no promotion of HCC using DAA treatment for HCV-related HCC patients.

Cetyltrimethylammonium Bromide Suppresses the Migration and Invasion of Hepatic Mahlavu Cells by Modulating Fibroblast Growth Factor Signaling.

Liver cancer is the fourth leading cause of cancer-related mortality globally, of which hepatocellular carcinoma (HCC) accounts for 85-90% of total primary liver cancer. A drug shortage for HCC therapy triggered us to screen the small-molecule database with a high-throughput cellular screening system. Herein, we examined whether cetyltrimethylammonium bromide (CTAB) inhibits cellular mobility and invasiveness of Mahlavu HCC cells. The effects of CTAB on cell viability were assessed using WST-1 assay, cell-cycle distribution using flow cytometric analysis, migration/invasion using woundhealing and transwell assays, and associated protein levels using western blotting. Treatment of Mahlavu cells with CTAB transformed its mesenchymal spindle-like morphology. In addition, CTAB exerted inhibitory effects on the migration and invasion of Mahlavu cells dose-dependently. CTAB also reduced the protein levels of matrix metalloproteinase-2 (MMP2), MMP9, RAC family small GTPase 1, SNAIL family transcriptional repressor 1 (SNAI1), SNAI2, TWIST family basic helix-loop-helix transcription factor 1 (TWIST1), vimentin, N-cadherin, phospho-fibroblast growth factor (FGF) receptor, phospho-phosphoinositide 3-kinase, phospho-v-Akt murine thymoma viral oncogene and phospho-signal transducer and activator of transcription 3 but increased the protein levels of tissue inhibitor of metalloproteinases-1/2 and E-cadherin. Rescue experiments proved that CTAB induced mesenchymal-epithelial transition in Mahlavu cells and this was significantly dose-dependently mitigated by basic FGF. CTAB suppressed the migration and invasion of Mahlavu cells through inhibition of the FGF signaling pathway. CTAB seems to be a potential agent for preventing metastasis of hepatic cancer.

Gaps in Study Design for Immune Parameter Research for Latent Tuberculosis Infection: A Systematic Review.

Background Immune parameters (IP) have been extensively studied to distinguish between latent tuberculosis (LTBI) and active tuberculosis (TB). Objective To determine the IP associated with LTBI, compared to active TB and individuals not infected by M. tuberculosis published in literature. Methods We conducted a systematic search using Google Scholar and PubMed databases, combining the MeSH terms latent tuberculosis, Mycobacterium tuberculosis, cytokines, and biological markers, with the free terms, biomarkers and cytokines. Spanish, English, and Portuguese articles comparing the concentration of IP associated with LTBI, either in plasma/serum or in vitro, in adults and nonimmunocompromised versus individuals with TB or without M. tuberculosis infection between 2006 July and 2018 July were included. Two blinded reviewers carried out the searches, read the abstracts, and selected the articles for analysis. Participants' information, diagnostic criteria, IP, detection methods, and biases were collected. Results We analyzed 36 articles (of 637 abstracts) with 93 different biomarkers in different samples. We found 24 parameters that were increased only in active TB (TGF-α, CSF3, CSF2, CCL1 [I-309], IL-7, TGF-β1, CCL3 [MIP-1α], sIL-2R, TNF-β, CCL7 [MCP-3], IFN-α, fractalkine, I-TAG, CCL8 [MCP-2], CCL21 [6Ckine], PDGF, IL-22, VEGF-A, LXA4, PGE2, PGF2α, sCD163, sCD14, and 15-Epi-LXA4), five were elevated in LTBI (IL-5, IL-17F, IL-1, CCL20 [MIP-3α], and ICAM-1), and two substances were increased among uninfected individuals (IL-23 and basic FGF). We found high heterogeneity between studies including failure to account for the time/illness of the individuals studied; varied samples and protocols; different clinical classification of TB; different laboratory methods for IP detection, which in turn leads to variable units of measurement and assay sensitivities; and selection bias regarding TST and booster effect. None of the studies adjusted the analysis for the effect of ethnicity. Conclusions It is mandatory to harmonize the study of immune parameters for LTBI diagnosis. This systematic review is registered with PROSPERO CRD42017073289.

Recombinant HvRNASET2 protein induces marked connective tissue remodelling in the invertebrate model Hirudo verbana.

The RNASET2 ribonuclease, belonging to the highly conserved RH/T2/s RNase gene family, has been recently shown to modulate inflammatory processes in both vertebrates and invertebrates. Indeed, the RNASET2 protein acts as a chemoattractor for macrophages in both in vitro and in vivo experimental settings and its expression significantly increases following bacterial infections. Moreover, we recently observed that injection of human recombinant RNASET2 protein in the body wall of the medicinal leech (a consolidated invertebrate model for both immune response and tissue regeneration) not only induced immune cell recruitment but also apparently triggered massive connective tissue remodelling as well. Based on these data, we evaluate here a possible role of leech recombinant RNASET2 protein (rHvRNASET2) in connective tissue remodelling by characterizing the cell types involved in this process through histochemical, morphological and immunofluorescent assays. Moreover, a time-course expression analysis of newly synthesized pro-collagen1α1 (COL1α1) and basic FGF receptor (bFGFR, a known fibroblast marker) following rHvRNASET2 injection in the leech body wall further supported the occurrence of rHvRNASET2-mediated matrix remodelling. Human MRC-5 fibroblast cells were also investigated in order to evaluate their pattern of collagen neosynthesis driven by rHvRNASET2 injection.Taken together, the data reported in this work provide compelling evidence in support of a pleiotropic role for RNASET2 in orchestrating an evolutionarily conserved crosstalk between inflammatory response and regenerative process, based on macrophage recruitment and fibroblast activation, coupled to a massive extracellular reorganization.

Alterations in the Levels of Growth Factors in Adolescents with Major Depressive Disorder: A Longitudinal Study during the Treatment with Fluoxetine.

Major depressive disorder (MDD) has a prevalence of 5% in adolescents. Several studies have described the association between the inflammatory response and MDD, but little is known about the relationship between MDD and growth factors, such as IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF. It must be appointed that there are scarce reports on growth factors in adolescents with MDD and even fewer with a clinical follow-up. In this work, we evaluated the levels of growth factors (IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF) in MDD adolescents and the clinical follow-up during eight weeks of treatment with fluoxetine. Methods. All patients were diagnosed according to the DSM-IV-TR, and the severity of the symptoms was evaluated using the Hamilton Depression Rating Scale (HDRS). Growth factors IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF were quantified by cytometric bead array using serum samples from 22 adolescents with MDD and 18 healthy volunteers. Results. All patients showed clinical improvement since the fourth week of pharmacological treatment according to the HDRS. Considerably higher levels of IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF were detected in MDD adolescents as compared to healthy volunteers. A significant but temporal decrease was detected in basic FGF, G-CSF, and GM-CSF at week four of fluoxetine administration. Conclusions. To the best of our knowledge, this is the first report to show alterations in the levels of growth factors, such as IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF in MDD adolescents during eight weeks of clinical follow-up. These disturbances might be involved in the physiopathology of MDD since such growth factors have been proven to participate in the neural development and correct functioning of the CNS; therefore, subtle alterations in it may contribute to MDD.

Tracheal cartilage growth promotion by intra-tracheal administration of basic FGF.

This study aimed to investigate whether intra-tracheal administration of basic fibroblast growth factor (b-FGF) promotes the growth of tracheal cartilage. Trachea of 4-week old mice were intubated and 2.5μg b-FGF administered (Group 4) for periods from 1 to 5days. Cervical tracheal outer diameter and tracheal ring length were compared in Group 1 (no intervention), Group 2 (tracheal intubation), Group 3 (intra-tracheal administration of distilled water) and Group 4, at 8weeks of age. Outer diameter and tracheal ring length in Group 4 were also compared with that in Group 1 at 12 and 16weeks of age. At 8weeks of age, tracheal ring length with b-FGF administration for more than 4days in Group 4 was significantly increased over that following 1-day administration. At 8weeks of age, mean outer diameter and the mean tracheal ring length in Group 4 were significantly greater than in the other groups. Mean outer diameter and mean tracheal ring length were significantly greater in Group 4 than in Group 1 at 12 and 16weeks of age. This study has shown that intra-tracheal administration of b-FGF enlarges the tracheal lumen.

Altered serum cytokine expression profile in systemic sclerosis and its regulatory mechanisms

To analyze the expression profile of serum cytokines in patients with systemic sclerosis (SSc) and explore its possible regulatory mechanisms. Serum and DNA of peripheral blood mononuclear cells were collected from 30 SSc patients and 80 normal controls (NCs). According to the presence or absence of interstitial lung disease (ILD) in SSc, the patients were divided into SSc with ILD group and SSc without ILD group. According to the degree of skin involvement, the patients were divided into diffuse systemic scleroderma (dcSSc) group and limited systemic scleroderma (lcSSc) group. According to the presence of anti-topoisomerase-1 antibody (anti-Scl-70 antibody) in the serum of patients with SSc, they were divided into SSc Scl-70 (+) group and SSc Scl-70 (-) group. 27 cytokines in serum were detected by Luminex MAGPIX detection system and Bio-Plex Pro Human Cytokine 27-plex Assay kit: interleukin-1β (IL-1β), interleukin-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12P70, IL-13, IL-15, IL-17, basic fiber growth factor (BASIC FGF), eotaxin, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-γ (IFN-γ), interferon-gamma induced protein 10(IP-10), monocyte chemotactic protein 1(MCP-1), macrophage inflammatory protein-1α (MIP-1α), macrophage inflammatory protein 1β(MIP-1β), platelet-derived growth factor BB (PDGF-BB), regulated on activation in normal T-cell expressed and secreted (RANTES), tumor necrosis factor-α (TNF-α), and vascular endothelial growth factor(VEGF). Methylation sites were detected by Illumina 450K methylation chip. Compared with NCs group, the expression of 12 cytokines (BASIC FGF, eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1β, IL-1ra, IL-6, IP-10, MCP-1, TNF-α and RANTES) in the SSc group significantly increased (P<0.05), IL-5 was decreased expression in the SSc group (P<0.05), there was no significant difference in the expressions of the other 14 cytokines. Compared with lcSSc group, 9 cytokines (eotaxin, IL-5, MCP-1, IL-2, RANTES, IL17A, IL-8, MIP-1β and PDGF-BB) increased in dcSSc group, but there was no significant difference. Compared with SSc without ILD group, IL-15 increased in SSC with ILD group [18.2 (172.97) ng/L vs. 2.03(0.05) ng/L, P<0.05]. Compared with SSc Scl-70 (-) group, the expression of IP-10 decreased in SSc Scl-70 (+) group [1 030 (2 196.6) ng/L vs. 1 878 (2 964) ng/L, P<0.05]. The correlation analysis of serum cytokines with erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) showed that IL-6 was positively correlated with ESR (r =0.04, P= 0.017), MCP-1 (r=0.49, P=0.043) and MIP-1β (r=0.41, P=0.007) positively correlated with CRP. By analyzing the changes of methylation sites of cytokines, it was found that cg17744604 in IL-10 TSS1500 region, cg06111286 in IL-12P70 TSS200 region, cg07935264 in IL-1β TSS200 region, cg01467417 in IL-1ra TSS1500 region, cg03989987 in IL-1ra 5'UTR region and cg21099624 in VEGF TSS200 region were all hypomethylated. There were different cytokines expression profiles in the serum of SSc patients, and the altered cytokines were correlected with the degree of skin damage and pulmonary fibrosis. Many cytokines were regulated by methylation.

Cytokine profiling reveals increased serum inflammatory cytokines in idiopathic choroidal neovascularization

BackgroundThe exact pathogenesis of idiopathic choroidal neovascularization (ICNV) remains unclear. Cytokine-mediated inflammation has been thought to be involved in the pathophysiology of ICNV. The purpose of this study was to investigate serum cytokine profiles in patients with ICNV and to explore the relationship between serum cytokine levels and ICNV severity.MethodsThis case-control study was conducted in 32 ICNV patients and 30 healthy volunteers. Clinical and demographic information was obtained from the medical data platform and the serum was analysed with a multiplex assay to determine the levels of seven cytokines: interleukin (IL)-2, IL-10, IL-15, IL-17, basic fibroblast growth factor (basic FGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and vascular endothelial growth factor (VEGF).ResultsSerum levels of IL-2, IL-10, IL-17, basic FGF, and VEGF were elevated in ICNV patients compared to controls. Serum GM-CSF levels were positively related to central retinal thickness, and serum IL-17 levels were positively related to CNV lesion area.ConclusionSerum inflammatory cytokines were significantly elevated in ICNV patients compared to controls. This suggests that systemic inflammation may play a critical role in the physiopathology of ICNV.

A Rabbit Corneal Endothelial Dysfunction Model Using Endothelial-Mesenchymal Transformed Cells

Unlike humans, rabbit corneal endothelial wounds are known to spontaneously heal. The current study was aimed to develop a new rabbit bullous keratopathy model using corneal endothelial cells that were induced to undergo endothelial-mesenchymal transformation (EMT). EMT was induced in rabbit corneal endothelial cells (RCECs) by culturing with TGFβ and basic FGF Supplemented Medium. The corneal endothelia in recipient rabbits were mechanically scraped from the corneal endothelial surface inside an 8 mm mark. Then, a suspension of EMT-induced RCECs (EMT-RCECs) was injected into the anterior chamber. Eyes injected with freshly isolated RCECs (Fresh RCECs group) and eyes that were scraped without injection of cells (Scrape group) were used as controls. Immediately following operation, subepithelial and stromal edema was observed with increased central corneal thickness and corneal opacity in all groups. In the EMT-RCECs group, bullous keratopathy persisted for 42 days up to the end of the study. In the Fresh-RCECs and Scrape groups, corneal transparency and thickness recovered by 7 days after treatment and was maintained up to 42 days. The activated fibroblast marker, α-SMA, was observed spanning from corneal endothelium to corneal stroma in the EMT-RCECs group. Interestingly, α-SMA was upregulated in the Scrape-group as well. In all groups, there was no damage to other intraocular structures, and intraocular pressure was normal throughout the observation period. Transplanting a fresh donor cornea effectively treated corneal edema due to bullous keratopathy. This model is a promising tool for pre-clinical trials in the development of new therapies against corneal endothelial dysfunction.

Expression of Halo-hFGF18 and study of its effect on differentiation of ATDC5 cells

Fibroblast growth factor 18 (FGF18) is a member of the fibroblast growth factor family and important in cartilage growth and development. However, the mechanism by which FGF18 mediates its biological functions is still unclear. In our study, we expressed the rhFGF18 protein fused to a HaloTag, (Halo-rhFGF18). MTT assay results indicated that both rhFGF18 and Halo-rhFGF18 have similar biological activities in NIH3T3 cells. However, basic FGF and acidic FGF were more potent than both rhFGF18 and Halo-rhFGF18. Confocal imaging data indicated that the red fluorescence labeled Halo-rhFGF18 strongly bound to ATDC5 cells and stimulated their proliferation and differentiation, which suggests that glycosaminoglycans may be involved in mediating the biological effects of rhFGF18 in ATDC5 cells. Moreover, western blot results demonstrated that, in ATDC5 cells, ERK1/2 signaling is activated upon stimulation with rhFGF18. Our results may open doors for the use of rhFGF18 as a drug to promote cartilage growth.

Distinct cytokine profiles in human brains resilient to Alzheimer's pathology

Our group has previously studied the brains of some unique individuals who are able to tolerate robust amounts of Alzheimer's pathological lesions (amyloid plaques and neurofibrillary tangles) without experiencing dementia while alive. These rare resilient cases do not demonstrate the patterns of neuronal/synaptic loss that are normally found in the brains of typical demented Alzheimer's patients. Moreover, they exhibit decreased astrocyte and microglial activation markers GFAP and CD68, suggesting that a suppressed neuroinflammatory response may be implicated in human brain resilience to Alzheimer's pathology. In the present work, we used a multiplexed immunoassay to profile a panel of 27 cytokines in the brains of controls, typical demented Alzheimer's cases, and two groups of resilient cases, which possessed pathology consistent with either high probability (HP, Braak stage V-VI and CERAD 2–3) or intermediate probability (IP, Braak state III-IV and CERAD 1–3) of Alzheimer's disease in the absence of dementia. We used a multivariate partial least squares regression approach to study differences in cytokine expression between resilient cases and both Alzheimer's and control cases. Our analysis identified distinct profiles of cytokines in the entorhinal cortex (one of the earliest and most severely affected brain regions in Alzheimer's disease) that are up-regulated in both HP and IP resilient cases relative to Alzheimer's and control cases. These cytokines, including IL-1β, IL-6, IL-13, and IL-4 in HP resilient cases and IL-6, IL-10, and IP-10 in IP resilient cases, delineate differential inflammatory activity in brains resilient to Alzheimer's pathology compared to Alzheimer's cases. Of note, these cytokines all have been associated with pathogen clearance and/or the resolution of inflammation. Moreover, our analysis in the superior temporal sulcus (a multimodal association cortex that consistently accumulates Alzheimer's pathology at later stages of the disease along with overt symptoms of dementia) revealed increased expression of neurotrophic factors, such as PDGF-bb and basic FGF in resilient compared to AD cases. The same region also had reduced expression of chemokines associated with microglial recruitment, including MCP-1 in HP resilient cases and MIP-1α in IP resilient cases compared to AD. Altogether, our data suggest that different patterns of cytokine expression exist in the brains of resilient and Alzheimer's cases, link these differences to reduced glial activation, increased neuronal survival and preserved cognition in resilient cases, and reveal specific cytokine targets that may prove relevant to the identification of novel mechanisms of brain resiliency to Alzheimer's pathology.

Osteogenic Extracellular Matrix Sheet and Its Healing Potential of Allogenic Implantation in a Rat Femoral Nonunion Model

Grant support received from: Japan Orthopaedics and Traumatology Research Foundation, Inc. and the Translational Research Network Program from the Japan Agency for Medical Research and Development, AMED There is no financial information to disclose. Minimally invasive percutaneous injection of osteogenic cell sheet (OCS) could increase bone regeneration in a rat nonunion model, but the allogenic implantation may have immunological rejection. We hypothesized that the osteogenic extracellular matrix sheet (OECMS), which has no cellular component, can retain growth factors integrity and enhance bone formation in a rat nonunion model. In an in vitro study, OCSs were fabricated from rat bone marrow stromal cells and OECMSs were devitalized from OCSs with liquid nitrogen. Bone morphogenetic protein 2 (BMP-2),vascular endothelial growth factors (VEGF)-A, and basic fibroblast growth factor (bFGF) concentrations in the OECMS were quantified using ELISA. In an in vivo study, we implanted OECMS in a rat femoral nonunion model. Eighteen male Fischer 344 inbred rats were divided into 2 groups, the OECMS group and the control group. Fracture healing was evaluated with radiological and histological analyses at 2, 5, and 8 weeks. Biomechanical analysis was performed at 8 weeks. Compared with the OCS group, cell viability of the OECMS group was 0.2%. BMP-2, VEGF-A, and bFGF was retained in the OECMS (BMP-2: OCS, 86 pg/ml; OECMS, 86 pg/ml, VEGF-A: OCS, 6,944 pg/ml; OECMS, 4,056 pg/ml, bFGF: OCS, 720 pg/ml; OECMS, 448 pg/ml) (Figure 74-1). Radiological evaluations demonstrated enhanced bone regeneration in the OECMS group compared with the control group at 8 weeks (union rate OECMS group: 57%, control group: 0%) . Histological evaluations demonstrated bridging callus over the fracture site in the OECMS group at 8 weeks (Figure 74-2). Biomechanical testing revealed a significantly higher maximum bending load and stiffness in the OECMS group compared with the control group. (OECMS, 45.2 ± 24 N; control, 10.3 ± 9.6 N; P < .05, OECMS, 116 ± 99 N/mm; control, 25.7 ± 21 N/mm; P < .05, respectively). •Cell viability of the OECMS group was significantly decreased compared with that of the OCS group.•OECMS contained rich BMP-2, VEGF-A, and basic FGF after the freeze-thaw cycling method using liquid nitrogen.•OECMS represents a novel sheet-shaped scaffold with bone healing potential and contains rich osteoinductive and osteoconductive ability.74-2Histological findings of fracture sites at 2, 5, and 8 weeks in osteogenic extracellular matrix sheet (OECMS) and control groups. At 2 weeks, large soft tissue covered the fracture site in the OECMS group; however, fibrous tissue was interpositioned between the ends of the fractured bone in the control group. At 5 weeks, the calcification was expanded onto the periosteum in the OECMS group. However, calcification was not confirmed over the fracture site in the control group. At 8 weeks after surgery, bridging callus over the fracture site was observed in the OECMS group. However, the control group showed fibrous granulation tissue at the fracture site, the ends of cortical bone had become rounded, and there was evidence of bony resorption.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Cycloartane triterpenoid (23R, 24E)-23-acetoxymangiferonic acid inhibited proliferation and migration in B16-F10 melanoma via MITF downregulation caused by inhibition of both \u03b2-catenin and c-Raf\u2013MEK1\u2013ERK signaling axis

We recently reported that (23R, 24E)-23-acetoxymangiferonic acid (23R-AMA), a cycloartane triterpenoid isolated by activity-guided separation from a methanol extract of Garcinia sp. bark, inhibited melanin production via inhibition of tyrosinase (TYR) expression in the B16-F10 melanoma cell line. Since 23R-AMA also inhibited microphthalmia-associated transcription factor (MITF) expression, an upstream factor of TYR, these features of 23R-AMA were thought to be appropriate for development of whitening cosmetics. However, 23R-AMA exhibited growth inhibition other than inhibition of melanin production in B16-F10 cells. Therefore, we investigated biological activities of 23R-AMA in detail, focused on its application as an anti-melanoma compound. In this study, we demonstrated that 23R-AMA inhibited cell proliferation and basic FGF (bFGF)-induced migration in B16-F10 cells. Furthermore, 23R-AMA promoted ser45/thr41 phosphorylation of β-catenin and suppressed its intranuclear accumulation, which was suggested to be related to inhibition of MITF expression. The transcriptional activity of MITF is known to be regulated by phosphorylation via activated ERK. Further investigation revealed that 23R-AMA inhibited phosphorylation of c-Raf, MEK-1, and ERK, and also that of upstream molecules including FAK and c-Src. These results suggested that 23R-AMA inhibited growth and migration of B16-F10 melanoma by regulating both MITF expression and its activity. The activities of 23R-AMA reported in this study are new aspects of cycloartane triterpenoids.

Cancer-Associated Fibroblasts Drive Glycolysis in a Targetable Signaling Loop Implicated in Head and Neck Squamous Cell Carcinoma Progression.

Despite aggressive therapies, head and neck squamous cell carcinoma (HNSCC) is associated with a less than 50% 5-year survival rate. Late-stage HNSCC frequently consists of up to 80% cancer-associated fibroblasts (CAF). We previously reported that CAF-secreted HGF facilitates HNSCC progression; however, very little is known about the role of CAFs in HNSCC metabolism. Here, we demonstrate that CAF-secreted HGF increases extracellular lactate levels in HNSCC via upregulation of glycolysis. CAF-secreted HGF induced basic FGF (bFGF) secretion from HNSCC. CAFs were more efficient than HNSCC in using lactate as a carbon source. HNSCC-secreted bFGF increased mitochondrial oxidative phosphorylation and HGF secretion from CAFs. Combined inhibition of c-Met and FGFR significantly inhibited CAF-induced HNSCC growth in vitro and in vivo (P < 0.001). Our cumulative findings underscore reciprocal signaling between CAF and HNSCC involving bFGF and HGF. This contributes to metabolic symbiosis and a targetable therapeutic axis involving c-Met and FGFR.Significance: HNSCC cancer cells and CAFs have a metabolic relationship where CAFs secrete HGF to induce a glycolytic switch in HNSCC cells and HNSCC cells secrete bFGF to promote lactate consumption by CAFs. Cancer Res; 78(14); 3769-82. ©2018 AACR.

Abstract LB-370: A novel ex vivo culture workflow to enrich and expand circulating tumor cells (CTCs) from patients with stage III/IV breast cancer (BCa)

Abstract Introduction: CTCs play a critical role in BCa metastases. Molecular and genomic characterization of CTCs may help to predict BCa prognosis and identify which patients may derive treatment benefit, especially for those with metastatic or recurrent disease. However, CTCs are present at very low concentrations in the peripheral blood and their enrichment and expansion is technically challenging. Here we describe an ex vivo CTC culture workflow to expand CTCs for patients with Stage III/IV BCa. Methods: Duplicate whole blood samples (7.5ml/each) were collected in EDTA tubes from 16 patients with stage III/IV BCa patients before or after systemic therapy and stored at 15-30 °C until processing. CTC enumeration was performed on one of the samples from each patient using the CELLSEARCH® System to confirm the presence of &amp;gt;=5 CTCs per 7.5mL of blood. CTCs enrichment was performed on the second sample from each patient using the ParsortixTM System (ANGLE PLC), a microfluidic based technology that captures rare cells based on size and deformability. Using a proprietary Cell Separation Cassette with a critical gap size of 6.5μm, the Parsortix System captured viable CTCs from the blood inside the cassette. The CTCs were then harvested by inverting the flow direction and flushing the CTCs from the cassette into 210μL of PBS. The harvested CTCs were transferred and grown in ultralow attachment plates containing RPMI-1640 medium supplemented with EGF (20ng/ml), basic FGF (20ng/ml), B27 (10ml), and 1×Antibiotic-antimycotic. CTCs were cultured in a humid 37oC incubator with 5% CO2 and 4% O2. CTC counting was performed every week using a hemocytometer. DNA isolation of the CTC culture was performed on Day 21 using DNAzol. Results: All 16 patients had &amp;gt;=5 CTCs as defined by the CELLSEARCH System (based on cellular morphology and the correct phenotype of CK+, EpCAM+, DAPI+ and CD45-) at the time of the blood collection. Using the Parsortix System, highly purified CTCs (ranging in number from 300 to 17,250 cells) were isolated from the blood samples for each of the 16 patients and placed into culture (day 0). During the first week, the CTCs could be expanded to 3.5 - 5.5 fold, and then to 9.5 - 22.5 fold during the second week, to maximum amount of 112,500 within 14 days. The isolated CTCs were maintained without altered morphology at the same concentrations until Day 21. An average of 186ng and 1200ng of DNA could be isolated and purified from ~10,000 and ~110,000 cultured CTCs, respectively. Conclusions: We identified and optimized a workflow for the recovery and culturing of CTCs from Stage III/IV BCa patients, demonstrating that cell-size dependent isolation using the Parsortix System allows for effective ex vivo culture. With further optimization, this strategy may be utilized for organoids development for drug testing and molecular analysis, representing an important tool for personalized precision therapy. Citation Format: Qiang Zhang, Youbin Zhang, Lisa Ellen Flaum, Brian Helfand, Lorenzo Gerratana, William Gradishar, Leonidas Platanias, Massimo Cristofanilli, Massimo Cristofanilli. A novel ex vivo culture workflow to enrich and expand circulating tumor cells (CTCs) from patients with stage III/IV breast cancer (BCa) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-370.

FGF-2 suppresses expression of nephronectin via JNK and PI3K pathways.

Nephronectin (Npnt), an extracellular matrix protein, is a ligand for integrin α8β1 and is involved in the development of various organs, such as the kidneys, bones, liver, and muscles. Previously, we found that Npnt expression was inhibited by various cytokines including transforming growth factor‐β (Tgf‐β) and oncostatin M (Osm). Fibroblast growth factor (Fgf)‐2, otherwise known as basic Fgf, also plays important roles in skeletal development and postnatal osteogenesis. In this study, Npnt expression was found to be suppressed by Fgf‐2 in MC3T3‐E1 cells, an osteoblast‐like cell line, in a dose‐ and time‐dependent manners. Furthermore, Fgf‐2‐mediated Npnt mRNA suppression was shown to involve the Jun N‐terminal kinase (JNK) and phosphoinositide‐3 kinase (PI3K) pathways. Together, our results suggest that FGF‐2 suppresses Npnt gene expression via JNK and PI3K pathways.

PSA-alpha-2-macroglobulin complex is enzymatically active in the serum of patients with advanced prostate cancer and can degrade circulating peptide hormones.

Prostate cancer cells produce high levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the tumor microenvironment but is presumed to be enzymatically inactive in the blood due to complex formation with serum protease inhibitors α-1-antichymotrypsin and α-2-macroglobulin (A2M). PSA-A2M complexes cannot be measured by standard ELISA assays and are also rapidly cleared from the circulation. Thus the exact magnitude of PSA production by prostate cancer cells is not easily measured. The PSA complexed to A2M is unable to cleave proteins but maintains the ability to cleave small peptide substrates. Thus, in advanced prostate cancer, sufficient PSA-A2M may be in circulation to effect total A2M levels, levels of cytokines bound to A2M and hydrolyze small circulating peptide hormones. Total A2M levels in men with advanced prostate cancer and PSA levels above 1000 ng/mL were measured by ELISA and compared to controls. Additional ELISA assays were used to measure levels of IL-6 and TGF-beta which can bind to A2M. The ability of PSA-A2M complexes to hydrolyze protein and peptide substrates was analyzed ± PSA inhibitor. Enzymatic activity of PSA-A2M in serum of men with high PSA levels was also assayed. Serum A2M levels are inversely correlated with PSA levels in men with advanced prostate cancer. Il-6 Levels are significantly elevated in men with PSA >1000 ng/mL compared to controls with PSA <0.1 ng/mL. PSA-A2M complex in serum of men with PSA levels >1000 ng/mL can hydrolyze small fluorescently labeled peptide substrates but not large proteins that are PSA substrates. PSA can hydrolyze small peptide hormones like PTHrP and osteocalcin. PSA complexed to A2M retains the ability to degrade PTHrP. In advanced prostate cancer with PSA levels >1000 ng/mL, sufficient PSA-A2M is present in circulation to produce enzymatic activity against circulating small peptide hormones. Sufficient PSA is produced in advanced prostate cancer to alter total A2M levels, which can potentially alter levels of a variety of growth factors such as IL-6, TGF-beta, basic FGF, and PDGF. Alterations in levels of these cytokines and proteolytic degradation of small peptide hormones may have profound effect on host-cancer interaction.

Spinal Cord Stimulation in Patients With Complex Regional Pain Syndrome: A Possible Target for Immunomodulation?

Complex regional pain syndrome (CRPS) is characterized by continued pain disproportional to the inciting event, sensory abnormalities, vasomotor and sudomotor disturbances, and motor and trophic changes. Inflammatory involvement has been demonstrated in past CRPS studies resulting in pain, swelling, and warmth. Currently, it is unknown whether spinal cord stimulation (SCS) has immunomodulatory properties. The aim of this study was to determine whether SCS has immunomodulatory properties in CRPS patients. The primary outcome parameters are cytokines (IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-15, IL-17, TNF-α, IFN-γ), chemokines (IP-10 and Eotaxin), and growth factors (VEGF, PDGFbb, and basic FGF) from interstitial fluid of artificial skin blisters before (T0-baseline without SCS) and after SCS therapy (T1-40 Hz standard frequency stimulation and T2-preferred frequency stimulation). Secondary outcome parameters were baseline demographics, CRPS signs, symptoms, and phenotype (inflammatory, vasomotor, dystonia, or neuropathic). Results were analyzed by means of a MANOVA repeated measures design. After SCS, the expression of both pro- and anti-inflammatory cytokines decreased over time in both the CRPS affected extremity and the contralateral extremity. The levels of IP-10, Eotaxin, VEGF, and PDGFbb were also significantly reduced bilaterally. There were no significant changes in IL-6 and TNF-α before and after SCS. The sensory signs, symptoms, and phenotype improved after SCS. SCS in CRPS patients attenuates T-cell activation, improves peripheral tissue oxygenation and decreases anti-angiogenetic activity which results in diminished endothelial dysfunction and improved bloodflow. The possible immunomodulatory effects of SCS opens new therapeutic possibilities in diseases with the involvement of the immune system and vasomotor disturbances, and requires further research on these mechanisms of action.

喉頭・気管における再生医学研究の進歩

Principles of tissue engineering include scaffold materials, stem cells and regulatory factors. With the availability of blood supply, an organ can be regenerated. Scaffold materials such as collagen, hydroxyapatite and polylactic acid, stem cells such as mesenchymal stem cells (MSC), adipose tissue derived stem cells (ASC) and induced pluripotent stem (iPS) cells, and regulatory factors such as basic FGF, IGF-1 and HGF have been utilized. Langer and Vacanti proposed “tissue engineering,” wherein they proposed creating tissues outside the body and then implanting them into the body; however, their implanted tissues became absorbed in the body in animal experiments. In contrast, our concept of “in situ tissue engineering” is designed to mediate the healing and tissue re-growth within the body itself.

Assessment of FGFR1 Over-Expression and Over-Activity in Lung Cancer Cells: A Toolkit for Anti-FGFR1 Drug Screening.

Lung cancer, caused mainly by smoking, is one of the most prevalent diseases in China, resulting in high mortality rates. The increasing incidence of chronic disease due to lung cancer places a huge burden on the welfare and cost to the Chinese society. Amplification of the fibroblast growth factor receptor 1 (FGFR1) is associated with high incidence and mortality in lung cancer patients. FGFR1 signaling is implicated in oncogenic traits such as proliferation, cell survival, angiogenesis, and migration. Targeting FGFR1 and its ligand basic FGF (bFGF) is a key step forward in developing new therapies for this crippling disease. Lung adenocarcinoma is the most common subtype of non-small-cell lung cancer. In this study, A549, a lung adenocarcinoma cell line widely used in vitro as a model for drug metabolism and as a transfection host, was used to study FGFR1. A stable lentiviral FGFR1 over-expression system in lung cancer cells is described for the study of anti-lung cancer drug candidates targeting FGFR1. Ligand binding to FGFR1 activates the PI3K/Akt/mTOR signaling pathway and increases adhesion, invasion, and migration in this model. Using a unique FGF monoclonal antibody developed in the laboratory, the overactive PI3K pathway was effectively blocked, abrogating the negative metastatic signaling pathways in lung cancer cells. Importantly, this model provides an effective and simple screening kit for anti-FGF1 drug compounds for lung cancer treatment and a tool for understanding the molecular mechanisms of the FGFR1 signaling pathway in lung cancer. Furthermore, this toolkit based on a FGFR1 lentiviral construct model is transferrable to study FGFR1 signaling in any type of cancer cell.