Effect Of Fibroblast Conditioned Medium Research Articles

Abstract TP140: Loss Of Fibroblasts Aggravates Hemorrhagic Brain Injury By Diminishing Timp2 Secretion

Although Col1a1 + fibroblast-like cells have been identified in large blood vessels in the CNS, their function remains largely unknown. To answer this important question, we generated mutant mice (Col1a1-DTR) with ablated fibroblast-like cells by crossing the Col1a1-Cre mice with DTR mice, which contain a loxP-flanked STOP sequence before diphtheria toxin receptor. The Col1a1-DTR mice were grossly normal after diphtheria toxin injection, suggesting that Col1a1 + fibroblast-like cells are dispensable under homeostatic conditions. After hemorrhagic stroke, however, these mice demonstrated exacerbated brain injury, including enhanced blood-brain barrier (BBB) leakage and aggravated injury volume. In addition, the expression of Collagen-1 and PDGFRα, two fibroblast markers, was significantly reduced. These findings highlight a beneficial role of Col1a1 + fibroblast-like cells in hemorrhagic stroke. Consistent with these results, similar results were also observed in vitro. Specifically, fibroblast-conditioned medium (FCM) attenuated endothelial damage (increased TEER and decreased FITC-Dextran leakage) in an in vitro model of hemorrhagic stroke. Subsequent mechanistic study revealed that FCM up-regulated the expression of various tight junction proteins (occludin, ZO-1, and claudin-5) without affecting that of caveolin-1, a key protein in transcytosis. These results indicate that fibroblasts protect BBB via paracellular rather than transcellular mechanism. To further investigate how exactly fibroblasts exert this beneficial role in hemorrhagic stroke, we performed mass spectrometry analysis, and identified TIMP2 (a protein with known BBB-protecting function) in the FCM. Using both pharmacological and genetic approaches, we further showed that ablating TIMP2 abolished the neuroprotective effect of FCM. Altogether, these findings suggest that Col1a1 + fibroblasts secrete TIMP2, which inhibits tight junction protein loss and paracellular leakage, promoting BBB repair and hemorrhagic stroke recovery. These results suggest that fibroblasts and TIMP2 may be targeted in the therapy of stroke.

Abstract 5099: Large-cell neuroendocrine carcinoma cells of the lung induce a tumor-promoting senescent phenotype in fibroblasts through MMP1 overexpression and TGFβ1

Abstract Tumor-associated fibroblasts (TAFs) exhibit an activated/fibrotic phenotype in all subtypes of non-small cell lung cancer. In contrast, we previously reported that lung TAFs exhibit premature senescence selectively in large cell neuroendocrine carcinoma of the lung (LCNEC), which is among the most aggressive subtypes of lung cancer. Moreover, we also reported that senescent fibroblasts enhance the growth and invasion of LCNEC cells in vitro and in vivo, and that the co-culture of LCNEC cells with normal fibroblasts was sufficient to induce fibroblast senescence. Intriguingly, whole-genome transcriptional profiling identified MMP1 as highly overexpressed in a panel of LCNEC cells versus non-LCNEC cell lines. Here we examined the role of MMP1 in LCNEC paracrine induction of fibroblast senescence. MMP-1 expression was silenced in LCNEC cancer cell lines by shRNA, and common senescent markers were analyzed after co-culture with normal fibroblasts, including β-galactosidase staining, cyclin-dependent kinase Inhibitor 2A expression (CDKN2A) and growth arrest. In addition, the tumor-promoting effects of fibroblast conditioned medium in LCNEC growth and invasion were measured. We confirmed that the LCNEC cell lines used in this study exhibited an increased expression of 3 neuroendocrine markers (CHGA, NCAM1 and SYP) compared to non-LCNEC cells. Induction of fibroblast senescence was confirmed after coculture with shScramble LCNEC cells. Moreover, knocking-down MMP1 in LCNEC cells was sufficient to abrogate fibroblast induced senescence upon co-culture, as well as the tumor-promoting traits of fibroblast's conditioned medium. The addition of active recombinant MMP1 (rMMP1) partially rescued the fibroblast senescent phenotype in co-culture with knocked-down MMP1 LCNEC cells, yet it was not sufficient to induce senescence when added to fibroblasts cultured alone. In contrast, treating fibroblasts with rMMP1 and the potent pro-fibrotic cytokine TGFβ1 was sufficient to induce both senescence and protumorigenic properties. Our results unveil a process of “niche construction” by LCNEC cells that is driven by their overexpression of MMP1, which induces senescence in adjacent fibroblasts that secrete factors that enhance the growth and invasion of LCNEC cells, thereby contributing to the aggressive nature of these tumors. In addition, our results reveal a new pathologic synergy between MMP1 and TGFβ1 in eliciting fibroblast senescence and enhancing its tumor-promoting traits. Moreover, our findings support that the aberrant carcinoma cell-fibroblast crosstalk mediated by MMP1 may be a suitable therapeutic target in LCNEC, which currently lacks targeted therapies. Citation Format: Marta Gabasa, Rafael Ikemori, Marselina Arshakyan, Evette Radisky, Noemí Reguard, Derek Radisky, Jordi Alcaraz. Large-cell neuroendocrine carcinoma cells of the lung induce a tumor-promoting senescent phenotype in fibroblasts through MMP1 overexpression and TGFβ1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5099.

P.2.b.055 - Changes in plasma concentrations of neurotrophin-4/5 and chemokine SDF-1 in depression

Salivary gland cells are surrounded by a complex stromal environment, in which fibroblasts are the main cells in proximity to the gland cells. In this study, the interaction between parotid gland acinar cells (PGACs), fibroblasts, and biomaterials was investigated. We prepared different biomaterials, including chitosan, polyvinyl alcohol (PVA), poly (ethylene-co-vinyl alcohol) (EVAL), polyvinylidene fluoride (PVDF), and tissue culture polystyrene (TCPS) to culture fibroblasts and then collect their conditioned media to culture PGACs. We observed no difference in AQP3, AQP5, and E-cadherin expression among different fibroblast conditioned medium treatments. Interestingly, α-amylase expression was obviously enhanced in PGACs cultured in the presence of conditioned medium from fibroblasts cultured on PVDF. Higher neurotrophin-4 (NT-4) expression was observed in PVDF-derived fibroblast conditioned medium using a growth factor protein array assay. In addition, directly adding NT-4 into the culture medium significantly promoted α-amylase expression by PGACs. Finally, nestin and βIII-tubulin expression by fibroblasts cultured on PVDF was also enhanced. Together, these results suggest that PVDF could promote α-amylase expression by PGACs via the NT-4 produced by fibroblasts.To date, there is no effective therapy for patients with dry mouth with persistent salivary hypofunction. The study made use of different biomaterials to culture fibroblasts and then collect their conditioned media to culture PGACs. It was found that the effect of fibroblast conditioned medium from PVDF on the α-amylase expression of PGACs was obviously enhanced and higher neurotrophin-4 (NT-4) expression was found in PVDF-derived fibroblast conditioned medium. In addition, directly adding NT-4 into the culture medium significantly promoted the expression of α-amylase by PGACs and the expression of nestin and βIII-tubulin of fibroblasts after being cultured on PVDF was enhanced. Therefore, the present study represents the first description of the role of NT-4 in the expression of α-amylase of PGACs and the role of PVDF in the reprogramming fibroblasts into neural progenitor-like cells, indicating that PVDF could promote the expression of α-amylase by PGACs via the NT-4 produced by fibroblasts.

Effects of biomaterial-derived fibroblast conditioned medium on the α-amylase expression of parotid gland acinar cells

Salivary gland cells are surrounded by a complex stromal environment, in which fibroblasts are the main cells in proximity to the gland cells. In this study, the interaction between parotid gland acinar cells (PGACs), fibroblasts, and biomaterials was investigated. We prepared different biomaterials, including chitosan, polyvinyl alcohol (PVA), poly (ethylene-co-vinyl alcohol) (EVAL), polyvinylidene fluoride (PVDF), and tissue culture polystyrene (TCPS) to culture fibroblasts and then collect their conditioned media to culture PGACs. We observed no difference in AQP3, AQP5, and E-cadherin expression among different fibroblast conditioned medium treatments. Interestingly, α-amylase expression was obviously enhanced in PGACs cultured in the presence of conditioned medium from fibroblasts cultured on PVDF. Higher neurotrophin-4 (NT-4) expression was observed in PVDF-derived fibroblast conditioned medium using a growth factor protein array assay. In addition, directly adding NT-4 into the culture medium significantly promoted α-amylase expression by PGACs. Finally, nestin and βIII-tubulin expression by fibroblasts cultured on PVDF was also enhanced. Together, these results suggest that PVDF could promote α-amylase expression by PGACs via the NT-4 produced by fibroblasts. Statement of SignificanceTo date, there is no effective therapy for patients with dry mouth with persistent salivary hypofunction. The study made use of different biomaterials to culture fibroblasts and then collect their conditioned media to culture PGACs. It was found that the effect of fibroblast conditioned medium from PVDF on the α-amylase expression of PGACs was obviously enhanced and higher neurotrophin-4 (NT-4) expression was found in PVDF-derived fibroblast conditioned medium. In addition, directly adding NT-4 into the culture medium significantly promoted the expression of α-amylase by PGACs and the expression of nestin and βIII-tubulin of fibroblasts after being cultured on PVDF was enhanced. Therefore, the present study represents the first description of the role of NT-4 in the expression of α-amylase of PGACs and the role of PVDF in the reprogramming fibroblasts into neural progenitor-like cells, indicating that PVDF could promote the expression of α-amylase by PGACs via the NT-4 produced by fibroblasts.

Scleroderma fibroblasts promote migration of mononuclear leucocytes across endothelial cell monolayers.

Perivascular infiltrates of inflammatory cells are a hallmark of lesional skin in scleroderma. We have explored the potential for scleroderma fibroblasts to modulate mononuclear leucocyte migration across endothelial cell monolayers in tissue culture, and to regulate expression of endothelial cell adhesion molecules. Fibroblasts were grown from skin biopsies of eight patients with active diffuse cutaneous scleroderma and from four healthy controls. Co-culture and conditioned medium transfer experiments examined the effect of soluble fibroblast products on mononuclear leucocyte (U937) cell migration across endothelial cell (1E-7) monolayers grown on tissue culture inserts. Co-culture of scleroderma, but not control fibroblasts, promoted transendothelial migration of U937 cells. Scleroderma fibroblast-conditioned medium had qualitatively similar effects and equivalent results were obtained using Jurkat-6 (T lymphocyte) cells, and with peripheral blood mononuclear cells from a patient with diffuse cutaneous scleroderma. Promotion of leucocyte migration does not appear to result from increased endothelial adhesion molecule expression, since fibroblast-conditioned medium did not up-regulate endothelial cell expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) or E-selectin. Moreover, leucocyte migration across cytokine-activated endothelial cell layers in co-culture with fibroblasts was less than across resting cells, although the selective effect of scleroderma fibroblast co-culture persisted. Recombinant monocyte chemoattractant protein-1 (MCP-1) or IL-8 increased passage of mononuclear leucocytes across endothelial cell monolayers, whilst anti-MCP-1, but not anti-IL-8 antibodies, significantly reduced the effect of fibroblast conditioned medium. These data suggest that systemic sclerosis (SSc) fibroblasts promote leucocyte migration across endothelial cell monolayers in tissue culture via an MCP-1-dependent mechanism. These findings may be relevant to the perivascular mononuclear leucocyte infiltrates characteristic of early SSc lesions.

Differential regulation of normal and tumoral breast epithelial cell growth by fibroblasts and 1,25-dihydroxyvitamin D3.

Mesenchymal-epithelial interactions are of paramount importance during normal and tumoral breast developments. We have investigated the paracrine growth regulation of normal and tumoral breast epithelial cells by fibroblasts derived from normal or pathological breast tissues. In some cases, breast cancer MCF-7 cells or normal epithelial cells in primary culture were cocultured with fibroblasts in a Transwell system allowing diffusible factor exchanges. Alternatively, conditioned medium produced by fibroblast cultures was added to epithelial cell cultures. Fibroblasts were shown to stimulate the proliferation of normal and carcinoma cells through paracrine mechanisms. However, the paracrine exchanges appeared to be different in normal versus tumoral breast epithelial cell growth regulation. Moreover, vitamin D-related compounds that have been proposed as anti-tumoral drugs were studied for their ability to affect normal and tumoral mammary epithelial cell proliferation and to interfere with the growth-regulatory activity of fibroblasts. Whereas vitamin D compounds inhibited MCF-7 cell growth, they led to a marked stimulation of the proliferation of normal mammary epithelial cells. Moreover, it was shown that the vitamin D analog EB 1089 can block the mitogenic effect of fibroblast-conditioned medium on tumoral but not normal breast epithelial cells. The differential effects of vitamin D compounds on cell proliferation provide further data in favor of the different behaviours of normal and tumoral mammary epithelial cells. The potential therapeutic use of vitamin D derivatives in the treatment of breast cancer is supported by these results but their growth-stimulatory properties on normal epithelial cells cannot be overlooked.

Hepatocyte growth factor stimulates extensive development of branching duct-like structures by cloned mammary gland epithelial cells.

Although epithelial-mesenchymal (stromal) interactions are thought to play an important role in embryonic and postnatal development of the mammary gland, the underlying mechanisms are still poorly understood. To address this issue, we assessed the effect of fibroblast-derived diffusible factors on the growth and morphogenetic properties of a clonally derived subpopulation (clone TAC-2) of normal murine mammary gland (NMuMG) epithelial cells embedded in collagen gels. Under control conditions, TAC-2 mammary gland epithelial cells suspended within collagen gels formed either irregularly shaped cell aggregates or short branching cord-like structures. Addition of conditioned medium from Swiss 3T3 or MRC-5 fibroblasts dramatically stimulated cord formation by TAC-2 cells, resulting in the development of an extensive, highly arborized system of duct-like structures, which in appropriate sections were seen to contain a central lumen. The effect of fibroblast conditioned medium was completely abrogated by antibodies against hepatocyte growth factor (also known as scatter factor), a fibroblast-derived polypeptide that we have previously shown induces tubulogenesis by Madin-Darby canine kidney epithelial cells. Addition of exogenous recombinant human hepatocyte growth factor to collagen gel cultures of TAC-2 cells mimicked the tubulogenic activity of fibroblast conditioned medium by stimulating formation of branching duct-like structures in a dose-dependent manner, with a maximal 77-fold increase in cord length at 20 ng/ml. The effect of either fibroblast conditioned medium or hepatocyte growth factor was markedly potentiated by the simultaneous addition of hydrocortisone (1 microgram/ml), which also enhanced lumen formation. These results demonstrate that hepatocyte growth factor promotes the formation of branching duct-like structures by mammary gland epithelial cells in vitro, and suggest that it may act as a mediator of the inducing effect of mesenchyme (or stroma) on mammary gland development.