Culture Supernatants Of Fibroblasts Research Articles

Metformin suppresses esophageal cancer progression through the radiation‑induced cellular senescence of cancer‑associated fibroblasts.

Senescent cells are known to secrete proteins, including inflammatory cytokines and damage‑associated molecular patterns. This phenomenon is known as the senescence‑associated secretory phenotype (SASP). SASP in cancer stromal fibroblasts is involved in cancer growth and progression. Conversely, metformin, an antidiabetic drug, has been reported to inhibit SASP induction by inhibiting the activation of NF‑κB, a regulator of SASP. To date, at least to the best of our knowledge, there have been no reports regarding cellular senescence in fibroblasts and tumor progression via the SASP‑mediated paracrine pathway. The present study thus aimed to elucidate the induction mechanisms of SASP in radiation‑induced fibroblasts and to determine its effects on cancer progression via the paracrine pathway. Furthermore, the present study aimed to determine whether controlling SASP using metformin suppresses cancer progression. A well‑differentiated esophageal cancer cell line established by the authors' department and fibroblasts isolated and cultured from the non‑cancerous esophageal mucosa of resected esophageal cancer cases were used for the experiments. Fibroblasts were irradiated with 8 Gy radiation, and the changes in the expression of the senescence markers, SA‑β‑gal, p21, p16 and NF‑κB were evaluated using immunofluorescent staining and western blot analysis in the presence or absence of metformin treatment. The culture supernatants of irradiated fibroblasts treated with metformin and those treated without metformin were collected and added to the cancer cells to evaluate their proliferative, invasive and migratory abilities. Vimentin and E‑cadherin expression levels were also evaluated using immunofluorescent staining and western blot analysis. The expression levels of p16, p21 and NF‑κB in irradiated fibroblasts were attenuated by treatment with metformin. Supernatants collected from irradiated fibroblasts exhibited the proliferative activity of esophageal cancer cells, and the promotion of migratory and invasion abilities, which may be due to epithelial‑mesenchymal transition and changes in cell morphology. These reactions were confirmed to be suppressed by the addition of the supernatant of cultured fibroblasts pre‑treated with metformin. On the whole, the present study demonstrates that fibroblasts in the cancer stroma may be involved in tumor progression through cellular senescence.

Exosomes derived from periodontitis induce hepatic steatosis through the SCD-1/AMPK signaling pathway

AimTo investigate the impact of exosomes released by Porphyromonas gingivalis-Lipopolysaccharide activated THP-1 macrophages and human periodontal ligament fibroblasts on hepatocyte fat metabolism. ResultsThe liver of rats with experimental periodontitis showed obvious steatosis and inflammation compared with control rats. The culture supernatant of macrophages and human periodontal ligament fibroblasts (hPDLFs), when stimulated with Pg-LPS, induced lipogenesis in HepG2 cells. Furthermore, the lipid-promoting effect was effectively inhibited by the addition of the exosome inhibitor GW4869. Subsequently, we isolated exosomes from cells associated with periodontitis. Exosomes released by Pg-LPS-stimulated macrophages and hPDLFs are taken up by hepatocytes, causing mRNA expression related to fat synthesis, promoting triglyceride synthesis, and aggravating NAFLD progression. Finally, two sets of exosomes were injected into mice through the tail vein. In vivo experiments have also demonstrated that periodontitis-associated exosomes promote the development of hepatic injury and steatosis, upregulate SCD-1 expression and inhibit the AMPK signaling pathway. ConclusionsIn conclusion, we found that exosomes associated with periodontitis promote hepatocyte adipogenesis by increasing the expression of SCD-1 and suppressing the AMPK pathway, which indicates that close monitoring of the progression of stomatopathy associated extra-oral disorders is important and establishes a theoretical foundation for the prevention and management of fatty liver disease linked to periodontitis.

Synovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP+). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP+ dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP+ pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.

#5108 SUSTAINED IL-6 SECRETION CAN LEAD TO AN AMPLIFIED INFLAMMATORY RESPONSE, MEDIATED BY THE ACTIVATION OF STAT3 AND NFKB VIA THE IL-6 AMPLIFIER LOOP

Abstract Background and Aims Organ transplants are the preferred treatment for end-stage organ failure. Potent and specific immunosuppressive agents have significantly decreased acute allograft rejection following renal transplantation. A significant barrier to long-term kidney allograft outcomes is chronic antibody-mediated rejection (CABMR). Chronic inflammation is a major cause of late graft loss that is mediated by soluble mediators released by immune and non-immune cells. IL-6 is a crucial cytokine that plays a central role in developing chronic inflammation. Non-immune cells, such as fibroblasts, have recently been identified as mediating chronic allograft rejection by activating the IL-6 amplifier loop (IL-6+IL-17). Our Aim of the study: -to study the effect of IL-6+IL-17 on secretion of IL-6 in the culture supernatant of fibroblasts derived from renal biopsy of chronic antibody mediated rejection (CABMR) patients; -to see the effect of anti-IL-6 (Tocilizumab)and anti-IL-17 on the secretion of IL-6 from the fibroblasts derived from kidney tissue of chronic antibody mediated rejection (CABMR) patients; and -to elucidate the pro-inflammatory pathway leading to increased IL-6 secretion by induction of Amplifier loop. Method Fibroblasts from grafted kidney from CABMR patients (n = 6) were cultured and stimulated with IL-6 (20ng/ µl), IL-17(50ng/ µl), IL-6 plus IL-17 for 24 hours. Levels of IL-6, MCP-1 and CCL20 were estimated in culture supernatants by ELISA as marker of IL-6 amplifier loop activation. mRNA expression of IL-6, MCP1, CCL20, and SOCS3 genes were measured in the stimulated fibroblasts. Stimulated Renal fibroblast cells from CABMR patients were lysed with Lysis buffer and subjected to SDS–PAGE and western blotting with anti-phospho-STAT3 and anti-phospho-NFκB p65. Additionally, IL-6, MCP1, and CCL20 levels were measured in Healthy control (n = 10), CABMR (n = 20), and non-CABMR (n = 30) patients. Results IL-6 and IL-17 synergistically induced more IL-6, CCL-20 & MCP-1 production from fibroblasts in culture supernatant. Gene expression analysis of IL-6, MCP1, and CCL20 was significantly higher with synergistic activation of IL-6 and IL-17 as compared to either IL-6 or IL-17 alone, while SOCS3 gene expression was downregulated. Our results also suggested that IL-6 Amplifier loop activation induces the NFκB and STAT3 signalling pathway activation in the non-immune cells like fibroblast derived from CABMR patients. Additionally, concentrations of IL-6, CCL-20 & MCP-1 in sera were significantly higher in CABMR patients compared to non-CABMR patients (p<0.001). There was a significant reduction in IL-6 concentration in culture supernatant with IL-6 and IL-17 inhibitor together and mRNA expression of IL-6, CCL20 and MCP-1 was significantly reduced while SOCS3 gene expression was upregulated. Conclusion In humans after kidney transplantation, IL-6 amplifier activation plays an active role in chronic rejection responses. Inhibition of IL-6 with Anti-IL-6 (Tocilizumab) and inhibition of IL-17 with Anti-IL-17 together reduces markers of tissue injury (IL-6, MCP1, CCL20) and rejection of allografts. so, IL-6 amplifier may be a therapeutic target for Chronic transplant rejection.

Tumor-derived exosomes elicit cancer-associated fibroblasts shaping inflammatory tumor microenvironment in head and neck squamous cell carcinoma

ObjectivesExosome-mediated reciprocal crosstalk between tumor and stromal cells plays a crucial role in tumor development and progression. This study investigated whether exosomes released from head and neck squamous cell carcinoma (HNSCC) tumor cells can convert normal fibroblasts into cancer-associated fibroblasts (CAF)-like cells and further analyzed the functional characterization of fibroblasts educated by tumor-derived exosomes. Materials and methodsExosomes secreted from HNSCC cell lines were isolated and normal fibroblasts were established from normal oropharyngeal mucosa. The effects of the exosomes on fibroblasts were examined by proliferation and migration assays, and exosome-educated fibroblasts were analyzed for the expression of eight genes (IL1B, IL6, CXCL8, TGFB1, ACTA2, FAP, CD274, and PDCD1LG2) by RT-qPCR. Moreover, T cells or CD14-positive cells were co-cultured with culture supernatants from exosome-educated fibroblasts. T-cell proliferation and macrophage polarization were examined using flow cytometry. Then, RNA sequencing (RNA-seq) of exosome-educated fibroblasts and the corresponding control fibroblasts was performed. ResultsTumor-derived exosomes enhanced fibroblast proliferation and migration. Moreover, gene expression analysis revealed upregulation of the gene expression of proinflammatory cytokines and immunoregulatory genes, and activated fibroblast marker genes. The culture supernatants of tumor-derived exosome-educated fibroblasts suppressed T cell proliferation and the induction of protumoral macrophages compared with those of control fibroblasts. Next, comprehensive RNA-seq analysis data revealed the activation of 11 signaling pathways, including IL-6- and IL-17-related signaling. ConclusionThese results indicate that HNSCC tumor cells induce and/or differentiate into CAFs through exosome-based cell-to-cell communication to create an inflammatory tumor microenvironment.

109 Fibroblast extracellular vesicles modulate keratinocyte differentiation during skin aging

Small extracellular vesicles (EVs), ie, microvesicles and exosomes, play different roles in skin homeostasis and functions. Here, we want to clarify the interactions between fibroblasts and keratinocytes through EVs during skin aging. Small EVs were purified by ultracentrifugation from monolayer culture supernatants of dermal fibroblasts derived from breast skin of young adult donors (18-20 years old). Both particles mean size of 100 nm (NTA analysis) and expression of CD63, CD81 and TSG-101 (western blotting) allowed us to confirm an enrichment of small EVs in the purified samples. Fibroblast small EVs were then used to treat child/adult-derived keratinocytes grown in monolayer culture and 3D reconstructed epidermis (RE). In monolayer culture, keratinocyte proliferation was not modified by fibroblast small EVs as shown by DNA quantification in a 72-hours study. However, EVs affected keratinocyte differentiation according to the age of keratinocyte donors. Indeed, in monolayer culture, an increase of expression of late differentiation markers (IVL, LOR, FLG, TGM1) was observed in keratinocytes from child donors. In contrast, a decrease of expression of both early (KRT1/10) and late differentiation markers (LOR, FLG) was seen in keratinocytes from adult donors. Such changes were also observed in RE treated by fibroblast small EVs and confirmed by KRT10, LOR and IVL expression using immunofluorescence analysis. Moreover, the granular layer thickness was respectively increased and decreased in RE cultured with keratinocytes derived from child and adult donors. Finally, the mRNA expression of transcription factors involved in the control of keratinocyte differentiation (RUNX1, KLF4, GATA3) was also modulated by fibroblast small EVs treatment. Taken together, our work shows that fibroblast small EVs modulate keratinocyte differentiation and suggests that aging can deeply modify keratinocyte responses to fibroblast small EVs.

Exosomal MicroRNA-21 Promotes Keloid Fibroblast Proliferation and Collagen Production by Inhibiting Smad7.

In keloid fibroblasts, microRNA-21 (miR-21) enhances activation of the TGF-β-Smad signaling pathway by down-regulating Smad7 expression, thereby promoting keloid fibroblast proliferation and collagen production. However, it is unclear whether miR-21 performs the above-mentioned functions through exosomal transport. Here, we extracted exosomes from the culture supernatants of keloid and normal skin fibroblasts and observed that both types of cells above secrete exosomes; however, keloid fibroblasts secreted significantly more exosomal miR-21 than normal skin fibroblasts (P < .001). Interestingly, we also observed that exosomal miR-21 could enter target keloid fibroblasts. In addition, inhibiting exosomal miR-21 up-regulated Smad7 protein expression and reduced Smad2 and Smad3 protein levels in target keloid fibroblasts. Furthermore, inhibiting exosomal miR-21 down-regulated collagen I and collagen III expression in target keloid fibroblasts, increased the proportion of apoptotic cells, and reduced cell proliferation. Taken together, these results show that exosomal miR-21 promoted proliferation and collagen production in keloid fibroblasts by inhibiting Smad7. Thus, we identified regulatory roles for miR-21 in promoting keloid fibroblast proliferation and participating in keloid formation and development. These findings imply that miR-21 may serve as a novel target for controlling the development of keloids.

Effect of tagitinin C isolated from kembang bulan [ Tithonia diversifolia (Hemsley) A. Gray] leaves on VEGF and TNF- α expressions of keloid fibroblast

Tagitinin C, an active constituent of Tithonia diversifolia (Hemsley) A. Gray, has been proven caninhibit the collagen deposition of keloid fibroblasts in vitro. However, its mechanism of action has not been widely studied. One possible mechanism involves growth factors and cytokines. Vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNF-α) play an important role in the collagen deposition. The study aimed to evaluate the effect of tagitinin C on VEGF and TNF-α expression in keloid fibroblasts culture. An experimental laboratory study using fibroblast cell lines at passages III and IV was performed. Treatments were divided into two groups i.e. the treatment groups after incubation with tagitinin Cat various concentration of 1, 0.5, 0.25, and 0.125μg/ mL for 72 h, and the control group using culture media without tagitinin C. Following after incubation, the VEGF and TNF-α levels of keloid fibroblast culture supernatant were measured by ELISA. Kruskal-Wallis test continued using Mann-Whitney test or one way Anova continued by independent t test were applied to evaluate the differences between groups. A p value of less than 0.05 was considered statistically significant. The VEGF levels significantly decreases in concentration-dependent manner after treatment of the tagitinin C at various concentrations (p&lt;0.05). However, no significantly difference in TNF-α levels was observed (p&gt; 0.05). In conclusion, tagitinin C decreases the VEGF expression of keloid fibroblasts. However, it has no effect on the TNF-α expression.

Generation of Marker-Free pbd-2 Knock-in Pigs Using the CRISPR/Cas9 and Cre/loxP Systems.

Porcine β-defensin 2 (PBD-2), expressed by different tissues of pigs, is a multifunctional cationic peptide with antimicrobial, immunomodulatory and growth-promoting abilities. As the latest generation of genome-editing tool, CRISPR/Cas9 system makes it possible to enhance the expression of PBD-2 in pigs by site-specific knock-in of pbd-2 gene into the pig genome. In this study, we aimed to generate marker-free pbd-2 knock-in pigs using the CRISPR/Cas9 and Cre/loxP systems. Two copies of pbd-2 gene linked by a T2A sequence were inserted into the porcine Rosa26 locus through CRISPR/Cas9-mediated homology-directed repair. The floxed selectable marker gene neoR, used for G418 screening of positive cell clones, was removed by cell-penetrating Cre recombinase with a recombination efficiency of 48.3%. Cloned piglets were produced via somatic cell nuclear transfer and correct insertion of pbd-2 genes was confirmed by PCR and Southern blot. Immunohistochemistry and immunofluorescence analyses indicated that expression levels of PBD-2 in different tissues of transgenic (TG) piglets were significantly higher than those of their wild-type (WT) littermates. Bactericidal assays demonstrated that there was a significant increase in the antimicrobial properties of the cell culture supernatants of porcine ear fibroblasts from the TG pigs in comparison to those from the WT pigs. Altogether, our study improved the protein expression level of PBD-2 in pigs by site-specific integration of pbd-2 into the pig genome, which not only provided an effective pig model to study the anti-infection mechanisms of PBD-2 but also a promising genetic material for the breeding of disease-resistant pigs.

Targeting LA7 breast cancer stem cells of rat through repressing the genes of stemness-related transcription factors using three different biological fluids

Down-regulation of stemness genes expression is important in differentiation therapy against cancer stem cells (CSCs). The aim of this study was to evaluate the Oct4 , Sox2, Nanog, and C-myc expression in rat breast cancer stem cells (LA7) which treated with human ovarian follicular fluid (FF), replicative senescent fibroblast culture supernatant (P14), and 16 h serum starved fibroblast supernatant (16 h-SFS). The cells were exposed to these biological fluids for 24 h, 72 h, and 7 days. Stem-loop RT-qPCR assay was used to quantify the expression of above mentioned genes. Results showed that FF had the least cytotoxic effect on the LA7 cells. Except for Nanog gene, exposure of LA7 cell line to 16 h-SFS and P14 decreased significantly expression of the three other genes after 24 h (P < 0.05). Nanog and Sox2 genes expression was also decreased in LA7 cells which have been already treated with FF for 24 h. Moreover, compared to the control solution, the expression of Oct4 increased significantly after 7 days exposure to FF (P < 0.05). Annexin V-PE /7-AAD-, acridine orange/ethidium bromide staining and doubling time assays revealed apoptosis and necrosis induction by these biological fluids in LA7 cells. Moreover, in an in vitro model of metastasis assay, i.e., scratch test, these fluids exhibited anti-LA7 migration activity which culminated in 16 h-SFS treated cells. Generally, this study showed that FF, 16 h-SFS, and P14 have positive effects on down-regulation of Nanog, Oct4, Sox2 and C-myc expression, and consequently can increase the differentiation of breast cancer stem cells. For the first time, this study provided some evidence indicating that some biological fluids have potential to differentiate the CSCs, show anti- survival, growth-, and cell migration activity.

Evaluation of the keratinocytes or fibroblasts culture supernatant in an inflammatory hyperalgesia model

ABSTRACT BACKGROUND AND OBJECTIVES: Inflammation is a defense response of the body to a cellular damage caused by physical, chemical or biological agents, which triggers, among other factors, pain. Although inflammation plays an important role in the protection and regeneration of tissue injury, inflammatory pain results in decreased quality of life. In view of this, the development of safe and less invasive forms for the treatment of inflammatory pain is of great importance. The objective of this study was to evaluate the antihyperalgesic potential of the culture supernatant of keratinocytes and human fibroblasts in an experimental model of inflammatory hyperalgesia. METHODS: Evaluation of carrageenan induced inflammatory hyperalgesia through the use of electronic von Frey in animal models treated with culture supernatant of keratinocytes and fibroblasts. RESULTS: Local administration of naloxone, a nonselective opioid antagonist, in peripheral tissue, has been observed to inhibit the antihyperalgesic effect of the keratinocyte culture supernatant. Fibroblast culture supernatant on days 1 and 3 reverses for 2 hours the carrageenan induced inflammatory hyperalgesia, which is mediated by µ opioid agonist. CONCLUSION: This study indicates that culture supernatant of fibroblasts and keratinocytes is capable of inducing antinociception in inflammatory hyperalgesia, mediated by the release of endogenous opioids. In addition, it has been observed that the analgesic effect of the fibroblast culture supernatant is mediated specifically by the µ opioid agonist, having a duration of 2 hours.

Systemic effects of starved fibroblast culture supernatant on immunosuppressed rats treated with cancer stem cells (LA7).

Background:The present study aimed to investigate and compare the effect of starved fibroblast culture supernatant (SFS), DMEM and normal saline alone or along with LA7 on dexamethasone-treated immunosuppressed Wistar rats.Methods:After the isolation of fibroblasts from the fresh foreskin of children, it was cultured in serum-free DMEM, and the supernatant collected after 16 hours (16h-SFS). This solution and the other treatments were injected subcutaneously into the rats from each group once daily for 14 days. The liver, intestine and lung histology along with blood cellular and biochemical characteristics were studied.Results:The results showed that dexamethasone as immunosuppressant reduced the body weight. The histological change in the liver was mild fibrosis induced by LA7+16h-SFS. Also, among the different blood cellular and biochemical indices measured, the eosinophil percentage in the 16h-SFS treated rats , glucose levels in the 16h-SFS+LA7 group and triglyceride concentrations in the 16h-SFS group were changed (p<0.05).Conclusion:This study showed that the secretions of starved fibroblasts especially that combined with LA7 cancer stem cells could induce some minor histological and biochemical changes in immunosuppressed rats, and also it opened a new window for subsequent investigations on unknown mechanisms related to this work.

Protective effect and related mechanisms of curcumin in rat experimental periodontitis

BackgroundCurcumin exhibits anti-inflammatory effects and has been suggested as a treatment for inflammatory diseases. The aim of this study was to investigate the effects of curcumin on the lipopolysaccharide induced inflammatory response in rat gingival fibroblasts in vitro and ligation-induced experimental periodontitis in vivo, and to speculate the possible anti-inflammatory mechanism of curcumin.MethodsThe gingival fibroblasts were incubated with different concentrations of curcumin in the absence or presence of lipopolysaccharide (LPS). Concentrations of interleukin-1β(IL-1β), tumor necrosis factor-α (TNF-α), osteoprotegerin (OPG) and soluble receptor activator of nuclear factor kappa-B ligand (RANKL) culture supernatants of rat gingival fibroblasts were determined by enzyme linked immunosorbent assay. The nuclear fraction of rat gingival fibroblasts was extracted and nuclear factor kappa-B (NF-κB) activation was assessed by western blotting to elucidate related mechanisms. Curcumin was given every two days by oral gavage. The gingival inflammation and alveolar bone loss between the first and second molars were observed by hematoxylin and eosin staining. Collagen fibers were observed by picro-sirius red staining. Alveolar bone loss was assessed by micro-CT analysis.ResultsCurcumin attenuated the production of IL-1β and TNF-α in rat gingival fibroblasts stimulated by LPS, and inhibited the LPS-induced decrease in OPG/sRANKL ratio and NF-κB activation. Curcumin significantly reduced gingival inflammation and modulated collagen fiber and alveolar bone loss in vivo.Conclusionscurcumin modulates inflammatory activity in rat periodontitis by inhibiting NF-κB activation and decreasing the OPG/sRANKL ratio induced by LPS.

ELTD1 Function in Hepatocellular Carcinoma is Carcinoma-Associated Fibroblast-Dependent

Introduction: EGF, latrophilin, and seven transmembrane domain containing 1 (ELTD1) constitutes an orphan G-protein-coupled receptor (GPCR) of the adhesion family. High expression of ELTD1 is correlated with favorable prognosis of hepatocellular carcinoma (HCC). After silencing ELTD1 expression, however, tumor invasiveness is drastically reduced. The underlying mechanism of this apparent contradictory phenomenon is unknown. Because adhesion GPCRs couple extracellular adhesion to intracellular signaling, as a member of this family, ELTD1 function may be related to its tumor microenvironment. We therefore investigated the interaction between ELTD1 and the HCC tumor microenvironment.Methods: ELTD1 expression was assessed by immunohistochemical analyses of tissue samples from two independent groups of 333 patients with HCC. Correlations between the ELTD1 expression and the clinicopathological values were examined. We also constructed ELTD1 overexpression and knockdown HCC cell lines and conducted a series of in vivo and in vitro ELTD1 functional assays. We further collected carcinoma associated fibroblast (CAF) culture supernatants to culture HCC cell lines and repeat the respective functional assays in comparison with the control group.Results: Clinicopathologic correlations and in vivo models indicated ELTD1 as a tumor suppressor gene, whereas in vitro experiments suggested that ELTD1 could promote malignancy in HCC cell lines. Immunohistochemical staining of the generated ELTD1 overexpression xenograft tumors demonstrated that the CAF markers vimentin and α-SMA were highly expressed compared to the control group. This suggests that ELTD1 expression is correlated to CAF distribution. In addition, culturing with CAF supernatants inhibited HCC cell proliferation and invasion rates, confirming the correlation between CAF and ELTD1.Conclusion: The results of this study indicated that ELTD1 regulation of HCC progression is CAF-dependent, suggesting that ELTD1 function is regulated by its tumor microenvironment. Further investigation is required to determine the underlying mechanisms.

Production of recombinant human procollagen type I C-terminal propeptide and establishment of a sandwich ELISA for quantification

Procollagen type I carboxy-terminal propeptide (PICP), derived from type I procollagen, has been identified as an indicator of type I collagen synthesis in bone matrix formation and skin recovery. PICP is a heterotrimeric glycoprotein consisting of two α1 chains (PICPα1) and one α2 chain (PICPα2). Here, we report the recombinant expression of human PICP using a mammalian expression system. Co-expression of PICPα1 and PICPα2 in HEK293F cells resulted in the production of functional PICP in the correctly assembled heterotrimeric form. Using the recombinant PICP as an antigen, we isolated PICP-specific human monoclonal antibodies from phage-displayed antibody libraries and raised rabbit polyclonal antibodies. Using those antibodies, we then developed a sandwich ELISA for PICP with a limit of detection of 1 ng/mL and a measurable range of 1–640 ng/mL. Both intra- and inter-assay imprecision values were <10%. For measuring PICP levels in human fibroblast cellular extracts and culture supernatants and a human serum, the developed ELISA kit displayed comparable performance to that of a commercialized kit. Our results provide an efficient production strategy for recombinant PICP, facilitating the generation of PICP-specific antibodies and development of PICP sandwich ELISA, with potential use in clinical diagnosis of serum samples and testing of cosmeceutical ingredients in fibroblast cell cultures.

The primary growth of laryngeal squamous cell carcinoma cells in vitro is effectively supported by paired cancer-associated fibroblasts alone.

Most primarily cultured laryngeal squamous cell carcinoma cells are difficult to propagate in vitro and have a low survival rate. However, in our previous work to establish a laryngeal squamous cell carcinoma cell line, we found that laryngeal cancer-associated fibroblasts appeared to strongly inhibit the apoptosis of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In this study, we investigated whether paired laryngeal cancer-associated fibroblasts alone can effectively support the growth of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In all, 29 laryngeal squamous cell carcinoma specimens were collected and primarily cultured. The laryngeal squamous cell carcinoma cells were separated from cancer-associated fibroblasts by differential trypsinization and continuously subcultured. Morphological changes of the cultured laryngeal squamous cell carcinoma cells were observed. Immunocytofluorescence was used to authenticate the identity of the cancer-associated fibroblasts and laryngeal squamous cell carcinoma cells. Flow cytometry was used to quantify the proportion of apoptotic cells. Western blot was used to detect the protein levels of caspase-3. Enzyme-linked immunosorbent assay was used to detect the levels of chemokine (C-X-C motif) ligand 12, chemokine (C-X-C motif) ligand 7, hepatocyte growth factor, and fibroblast growth factor 1 in the supernatants of the laryngeal squamous cell carcinoma and control cells. AMD3100 (a chemokine (C-X-C motif) receptor 4 antagonist) and an anti-chemokine (C-X-C motif) ligand 7 antibody were used to block the tumor-supporting capacity of cancer-associated fibroblasts. Significant apoptotic changes were detected in the morphology of laryngeal squamous cell carcinoma cells detached from cancer-associated fibroblasts. The percentage of apoptotic laryngeal squamous cell carcinoma cells and the protein levels of caspase-3 increased gradually in subsequent subcultures. In contrast, no significant differences in the proliferation capacity of laryngeal squamous cell carcinoma cells cocultured with cancer-associated fibroblasts were detected during subculturing. High level of chemokine (C-X-C motif) ligand 12 was detected in the culture supernatant of cancer-associated fibroblasts. The tumor-supporting effect of cancer-associated fibroblasts was significantly inhibited by AMD3100. Our findings demonstrate that the paired laryngeal cancer-associated fibroblasts alone are sufficient to support the primary growth of laryngeal squamous cell carcinoma cells in vitro and that the chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 axis is one of the major contributors.

Effects of silencing Smad ubiquitination regulatory factor 2 on the function of human hypertrophic scar-derived fibroblasts

Effects of silencing Smad ubiquitination regulatory factor 2 on the function of human hypertrophic scar-derived fibroblasts

Abstract 227: Leptin induces a fibroblast and endothelial cell crosstalk in tumor stroma

Abstract Background: The tissue microenvironment plays a major role in tumor development and recurrence. It is known that leptin secreted by cancer and adipose cells can modify the tumor stroma, which is composed of immune, fibroblast, and endothelial cells (EC). Leptin affects both normal and cancer associated fibroblasts. We have previously shown that leptin can modify EC directly via an increase of angiogenic features; which was linked to VEGFR signaling and Notch expression independent of VEGF. It is hypothesized that leptin induces a communication between fibroblast and endothelial cells via induction of Notch signaling, which may be related to exosome production. Methods: Non-expressing Ob-R fibroblasts, and fibroblasts transfected with Ob-R plasmid, were used to investigate leptin's actions on Notch activation. Fibroblasts were challenged with leptin (0.6, 1.2, 6.2 nM) and an inhibitor of Notch signaling activation (DAPT). Fibroblast culture supernatants were added to and human umbilical vein endothelial cells (HUVEC) cultures. Notch proteins and RNA expression and molecular markers of activated fibroblasts and derived exosomes were determined via Western blot, RT-PCR, and multiplex analyses, respectively. EC angiogenic features were also determined. Results: Our findings suggest that leptin induces fibroblast activation, which triggers the expression of Notch and the development of angiogenic features by EC. Conclusions: A novel mechanism is proposed by which leptin promotes tumor stroma development via Notch activation and a crosstalk between fibroblasts and EC. Cancer therapies targeting leptin signaling could be a new strategy for interfering with cellular processes that promotes changes of tumor stroma towards a more aggressive tumor environment. Citation Format: Viola Lanier. Leptin induces a fibroblast and endothelial cell crosstalk in tumor stroma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 227.

The role of HGF/MET and FGF/FGFR in fibroblast-derived growth stimulation and lapatinib-resistance of esophageal squamous cell carcinoma.

BackgroundAlthough advanced esophageal squamous-cell carcinoma (ESCC) is treated using a multidisciplinary approach, outcomes remain unsatisfactory. The microenvironment of cancer cells has recently been shown to strongly influence the biologic properties of malignancies. We explored the effect of supernatant from esophageal fibroblasts on the cell growth and chemo-resistance of ESCC cell lines.MethodsWe used 22 ESCC cell lines, isolated primary human esophageal fibroblasts and immortalized fibroblasts. We first examined cell proliferation induced by fibroblast supernatant. The effect of supernatant was evaluated to determine whether paracrine signaling induced by fibroblasts can influence the proliferation of cancer cells. Next, we examined the effects of adding growth factors HGF, FGF1, FGF7, and FGF10, to the culture medium of cancer cells. These growth factors are assumed to be present in the culture supernatants of fibroblasts and may exert a paracrine effect on the proliferation of cancer cells. We also examined the intrinsic role of HGF/MET and FGFs/FGFR in ESCC proliferation. In addition, we examined the inhibitory effect of lapatinib on ESCC cell lines and studied whether the fibroblast supernatants affect the inhibitory effect of lapatinib on ESCC cell proliferation. Finally, we tested whether the FGFR inhibitor PD-173074 could eliminate the rescue effect against lapatinib that was induced by fibroblast supernatants.ResultsThe addition of fibroblast supernatant induces cell proliferation in the majority of cell lines tested. The results of experiments to evaluate the effects of adding growth factors and kinase inhibitors suggests that the stimulating effect of fibroblasts was attributable in part to HGF/MET or FGF/FGFR. The results also indicate diversity in the degree of dependence on HGF/MET and FGF/FGFR among the cell lines. Though lapanitib at 1 μM inhibits cell proliferation by more than 50% in the majority of the ESCC cell lines, fibroblast supernatant can rescue the growth inhibition of ESCC cells. However, the rescue effect is abrogated by co-treatment with FGFR inhibitor.ConclusionThese results demonstrate that cell growth of ESCC depends on diverse receptor tyrosine kinase signaling, in both cell-autonomous and cell-non-autonomous manners. The combined inhibition of these signals may hold promise for the treatment of ESCC.

Biocompatibility of three bioabsorbable membranes assessed in FGH fibroblasts and human osteoblast like cells culture.

ObjectivesSpecific physical and chemical features of the membranes may influence the healing of periodontal tissues after guided tissue regeneration (GTR). The aim of the present investigation was to analyze the biological effects of three bioabsorbable membranes. The hypothesis is that all tested membranes present similar biological effects.MethodsHuman osteoblast like-cells (SaOs-2) and gingival fibroblasts FGH (BCRJ -RJ) were cultured in DMEM medium. The viability of the cells cultured on the membranes was assesses using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Quantitative determination of activated human Transforming Growth Factor beta 1 (TGF-β1) on the supernatants of the cell culture was observed. Samples were examined using scanning electron microscope (SEM).ResultsSaOs2, in 24 hours, PLA group showed higher values when compared to other groups (P < 0.05). All groups presented statistical significance values when compared two times. In 4 h and 24 h, for the fibroblasts group, significantly difference was found to PLA membrane, when compared with the other groups (p < 0.05). For TGFβ1 analyzes, comparing 4 and 24 h, for the osteoblast supernatant, COL1 and PLA groups showed statistically significant difference (p <0,008). On the analysis of culture supernatants of fibroblasts, in 24 hours, only PLA group presented significant difference (p = 0,008).ConclusionsThe biomaterials analyzed did not show cytotoxicity, since no membrane presented lower results than the control group. PLA membrane presented the best performance due to its higher cell viability and absorbance levels of proliferation. Both collagen membranes showed similar results either when compared to each other or to the control group.