Absence Of Fibroblasts Research Articles

Proteolytic remodeling of 3D bioprinted tumor microenvironments

In native tissue, remodeling of the pericellular space is essential for cellular activities and is mediated by tightly regulated proteases. Protease activity is dysregulated in many diseases, including many forms of cancer. Increased proteolytic activity is directly linked to tumor invasion into stroma, metastasis, and angiogenesis as well as all other hallmarks of cancer. Here we show a strategy for 3D bioprinting of breast cancer models using well-defined protease degradable hydrogels that can facilitate exploration of the multifaceted roles of proteolytic extracellular matrix remodeling in tumor progression. We designed a set of bicyclo[6.1.0]nonyne functionalized hyaluronan (HA)-based bioinks cross-linked by azide-modified poly(ethylene glycol) (PEG) or matrix metalloproteinase (MMP) degradable azide-functionalized peptides. Bioprinted structures combining PEG and peptide-based hydrogels were proteolytically degraded with spatial selectivity, leaving non-degradable features intact. Bioprinting of tumor-mimicking microenvironments using bioinks comprising human breast cancer cells (MCF-7) and fibroblast in hydrogels with different susceptibilities to proteolytic degradation shows that MCF-7 proliferation and spheroid size were significantly increased in protease degradable hydrogel compartments, but only in the presence of fibroblasts. In the absence of fibroblasts in the stromal compartment, cancer cell proliferation was reduced and did not differ between degradable and nondegradable hydrogels. The interactions between spatially separated fibroblasts and MCF-7 cells consequently resulted in protease-mediated remodeling of the bioprinted structures and a significant increase in cancer cell spheroid size, highlighting the close interplay between cancer cells and stromal cells in the tumor microenvironment and the influence of proteases in tumor progression.

Stability of the Arginine-Ornithine-Citrulline Cycle Maintained by Tumor-Stroma Interactions in Cell Coculture Hydrogel Microspheres.

Tumor-stroma interaction is the core process of tumor metastasis. Therefore, building a model of tumor-stromal cell communication is crucial for understanding the tumor metastasis process and curing cancer completely. In this research, a controllable three-dimensional (3D) tumor-stroma coculture microsphere model based on droplet microfluidic technology was developed to culture human lung cancer cells (A549 cell) and fibroblast cells (NIH-3T3 cell) using core-shell hydrogel microspheres to partition different kinds of cells. In our coculture model, tumor cells show a trend of epithelial-mesenchymal transition (EMT): a decrease in the number of surface E-cadherin and an increase in the number of N-cadherin. At the same time, fibroblasts are activated into cancer-associated fibroblasts (CAFs) as the level of interleukin-6 (IL-6) released is increased. In addition, an interesting phenomenon was discovered; in the absence of fibroblasts, the metabolism of the tumor cell culture alone leads to arginine depletion and citrulline accumulation, whereas a coculture can maintain the arginine-ornithine-citrulline cycle to reach equilibrium after 72 h, and the balance increases the stress resistance of tumor cells. This discovery may provide a new direction for understanding tumor resistance.

Fibroblasts Impair Migration and Antitumor Activity of NK-92 Lymphocytes in a Melanoma-on-Chip Model.

Adoptive cell therapy in solid tumors, such as melanoma, is impaired, but little is known about the role that the fibroblasts present in the tumor microenvironment could exert. However, the mechanism at play is not well understood, partly due to the lack of relevant pre-clinical models. Three-dimensional culture and microfluidic chips are used to recapitulate the dynamic interactions among different types of cells in the tumor microenvironment in controlled and physiological settings. In this brief report, we propose a reductionist melanoma-on-a-chip model for evaluating the essential role of fibroblasts in the antitumor activity of lymphocytes. To this end, 3D melanoma spheroids were monocultured and co-cultured with human dermal fibroblasts and the NK-92 cell migration towards the tumor compartment was tested in a commercially available microfluidic device. Utilizing confocal microscopy, we observed the different recruitment of NK-92 cells in the presence and absence of fibroblasts. Our results show that fibroblasts' presence inhibits immune effector recruiting by exploiting a 3D pre-clinical tumor model.

A Transwell-Based Vascularized Model to Investigate the Effect of Interstitial Flow on Vasculogenesis.

Interstitial flow plays a significant role in vascular system development, mainly including angiogenesis and vasculogenesis. However, compared to angiogenesis, the effect of interstitial flow on vasculogenesis is less explored. Current in vitro models for investigating the effect of interstitial flow on vasculogenesis heavily rely on microfluidic chips, which require microfluidic expertise and facilities, and may not be accessible to biological labs. Here, we proposed a facile approach to building perfusable vascular networks through the self-assembly of endothelial cells in a modified transwell format and investigated the effect of interstitial flow on vasculogenesis. We found that the effect of interstitial flow on vasculogenesis was closely related to the existence of VEGF and fibroblasts in the developed model: (1) In the presence of fibroblasts, interstitial flow (within the range of 0.1-0.6 μm/s) facilitated the perfusability of the engineered vasculatures. Additional VEGF in the culture medium further worked synergically with interstitial flow to develop longer, wider, denser, and more perfusable vasculatures than static counterparts; (2) In the absence of fibroblasts, vasculatures underwent severe regression within 7 days under static conditions. However, interstitial flow greatly inhibited vessel regression and enhanced vascular perfusability and morphogenesis without the need for additional VEGF. These results revealed that the effect of interstitial flow might vary depending on the existence of VEGF and fibroblasts, and would provide some guidelines for constructing in vitro self-assembled vasculatures. The established transwell-based vascularized model provides a simple method to build perfusable vasculatures and could also be utilized for creating functional tissues in regenerative medicine.

Agent-Based Models Help Interpret Patterns of Clinical Drug Resistance by Contextualizing Competition Between Distinct Drug Failure Modes

Modern targeted cancer therapies are carefully crafted small molecules. These exquisite technologies exhibit an astonishing diversity of observed failure modes (drug resistance mechanisms) in the clinic. This diversity is surprising because back of the envelope calculations and classic modeling results in evolutionary dynamics suggest that the diversity in the modes of clinical drug resistance should be considerably smaller than what is observed. These same calculations suggest that the outgrowth of strong pre-existing genetic resistance mutations within a tumor should be ubiquitous.Yet, clinically relevantdrug resistance occurs in the absence of obvious resistance conferring genetic alterations. Quantitatively, understanding the underlying biological mechanisms of failure mode diversity may improve the next generation of targeted anticancer therapies. It also provides insights into how intratumoral heterogeneity might shape interpatient diversity during clinical relapse. We employed spatial agent-based models to explore regimes where spatial constraintsenable wild type cells (that encounter beneficial microenvironments)tocompete against genetically resistant subclones in the presence of therapy. In order to parameterize a model of microenvironmental resistance, BT20 cells were cultured in the presence and absence of fibroblasts from 16different tissues. The degree of resistance conferred by cancer associated fibroblasts in the tumor microenvironment was quantified by treating mono- and co-cultures with letrozole and then measuring the death rates. Our simulations indicate that, even when a mutation is more drug resistant, its outgrowth can be delayed byabundant,low magnitude microenvironmental resistance across large regions of a tumor that lack genetic resistance.These observations hold fordifferent modes of microenvironmental resistance, including juxtacrine signaling, soluble secreted factors,and remodeled ECM.This result helps to explain the remarkable diversity of resistance mechanisms observed in solid tumors, which subverts the presumption that the failure mode that causes the quantitatively fastest growth in the presence of drug should occur most often in the clinic. Our model results demonstrate that spatial effects can interact with low magnitude of resistance microenvironmental effects to successfully compete against genetic resistance that is orders of magnitude larger. Clinical outcomes of solid tumors are intrinsically connected to their spatial structure, and the tractability of spatial agent-based models like the ones presented here enable us to understand this relationship more completely.

Regulation of extracellular matrix composition by fibroblasts during perinatal cardiac maturation

BackgroundCardiac fibroblasts are the main non-myocyte population responsible for extracellular matrix (ECM) production. During perinatal development, fibroblast expansion coincides with the transition from hyperplastic to hypertrophic myocardial growth. Therefore, we investigated the consequences of fibroblast loss at the time of cardiomyocyte maturation by depleting fibroblasts in the perinatal mouse. Methods and resultsWe evaluated the microenvironment of the perinatal heart in the absence of fibroblasts and the potential functional impact of fibroblast loss in regulation of cardiomyocyte cell cycle arrest and binucleation. Cre-mediated expression of diphtheria toxin A in PDGFRα expressing cells immediately after birth eliminated 70–80% of the cardiac fibroblasts. At postnatal day 5, hearts lacking fibroblasts appeared similar to controls with normal morphology and comparable numbers of endothelial and smooth muscle cells, despite a pronounced reduction in fibrillar collagen. Immunoblotting and proteomic analysis of control and fibroblast-deficient hearts identified differential abundance of several ECM proteins. In addition, fibroblast loss decreased tissue stiffness and resulted in increased cardiomyocyte mitotic index, DNA synthesis, and cytokinesis. Moreover, decellularized matrix from fibroblast-deficient hearts promoted cardiomyocyte DNA replication. While cardiac architecture was not overtly affected by fibroblast reduction, few pups survived past postnatal day 11, suggesting an overall requirement for PDGFRα expressing fibroblasts. ConclusionsThese studies demonstrate the key role of fibroblasts in matrix production and cardiomyocyte cross-talk during mouse perinatal heart maturation and revealed that fibroblast-derived ECM may modulate cardiomyocyte maturation in vivo. Neonatal depletion of fibroblasts demonstrated that although hearts can tolerate reduced ECM composition, fibroblast loss eventually leads to perinatal death as the approach simultaneously reduced fibroblast populations in other organs.

Dermal Pericytes Exhibit Declined Ability to Promote Human Skin Regeneration with Ageing in 3D Organotypic Culture Models.

The well documented decline in the regenerative ability of ageing human skin has been attributed to many factors including genomic instability, telomere shortening, poor nutrient sensing, cellular senescence, and stem cell exhaustion. However, a role for the dermal cellular and molecular microenvironment in skin ageing is just emerging. We previously showed that dermal pericytes co-operate with fibroblasts to improve human skin regeneration in an organotypic skin culture model, and even do so in the absence of fibroblasts. Here, we report that the number of dermal cells, particularly pericytes, declines significantly in human skin of donors aged > 50 years. Notably, aged pericytes promoted epidermal regeneration of neonatal keratinocytes in organotypic cultures and the resulting epithelium exhibited a Ki67+/ΔNp63+ basal layer and terminal differentiation. However, the epithelium lacked several features of homeostasis displaying lower levels of ΔNp63 expression, decreased LAMA5 deposition at the dermo-epidermal junction, and the absence of basement membrane and hemi-desmosome assembly. We conclude that a decline in pericyte incidence and function contribute to an impaired epidermal microenvironment and poor skin regeneration with ageing in the human skin.

Cardiac Fibroblast Regulation of Matrix Assembly and Inflammation

Heart failure is a progressive disease that is often exacerbated by inappropriate cardiac remodeling in response to stress. The excess deposition of extracellular matrix (ECM) by fibroblasts, known as fibrosis, is often coincident with reduced cardiac output . While the detrimental effects of overactive ECM secretion are appreciated, our current understanding of the function of the cardiac fibroblast in the absence of stress is limited. Using a genetic approach in mice to deplete hearts of cardiac fibroblasts, we have investigated how loss of fibroblasts affects heart morphogenesis in the neonate and the adult. In the absence of fibroblasts at postnatal day 5, we observe limited production of ECM especially type I collagen. In the absence of ECM, cardiomyocytes bypass the growth arrest that usually occurs in the first week of postnatal growth and continue to undergo cytokinesis. These data suggest that ECM content may restrict cardiomyocyte growth and loss of ECM may be permissive for continued cardiomyocyte proliferation. To investigate the role of fibroblasts in heart homeostasis and inflammation, we also depleted cardiac fibroblasts from adult hearts. Although subtle alterations in cardiac structure were observed, hearts with 70–80% reduction in fibroblasts were functionally indistinguishable from controls suggesting that type I collagen is more stable than previously thought. Finally, we will describe how the loss of fibroblasts impacts inflammatory responses after injury. While an inflammatory response after acute injury is essential for clearing dead tissue, and some fibrosis may be necessary to maintain the tensile strength of injured myocardium, a sustained inflammatory response can lead to inappropriate fibroblast activation. After injury we observed increased cardiomyocyte hypertrophy and reduced infiltration of neutrophils, monocytes, and T and B cells. Cardiac fibroblast specific translatome analysis revealed that chemokine expression was dynamic after myocardial infarction. In the first four days after injury, Cxcl4, Cxcl10, and Ccl5 were increased, while Ccl17 expression increased and Ccl11 decreased after 8 days of injury. Our results suggest that fibroblasts are essential for ECM production and fibroblast depletion leads to changes in cardiomyocyte biology and inflammation after injury.Support or Funding InformationNHLBI Grant HL144067AHA 17GRNT33660474This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Collagen abundance controls melanoma phenotypes through lineage-specific microenvironment sensing

Despite the general focus on an invasive and de-differentiated phenotype as main driver of cancer metastasis, in melanoma patients many metastatic lesions display a high degree of pigmentation, indicative for a differentiated phenotype. Indeed, studies in mice and fish show that melanoma cells switch to a differentiated phenotype at secondary sites, possibly because in melanoma differentiation is closely linked to proliferation through the lineage-specific transcriptional master regulator MITF. Importantly, while a lot of effort has gone into identifying factors that induce the de-differentiated/invasive phenotype, it is not well understood how the switch to the differentiated/proliferative phenotype is controlled. We identify collagen as a contributor to this switch. We demonstrate that collagen stiffness induces melanoma differentiation through a YAP/PAX3/MITF axis and show that in melanoma patients increased collagen abundance correlates with nuclear YAP localization. However, the interrogation of large patient datasets revealed that in the context of the tumour microenvironment, YAP function is more complex. In the absence of fibroblasts, YAP/PAX3-mediated transcription prevails, but in the presence of fibroblasts tumour growth factor-β suppresses YAP/PAX3-mediated MITF expression and induces YAP/TEAD/SMAD-driven transcription and a de-differentiated phenotype. Intriguingly, while high collagen expression is correlated with poorer patient survival, the worst prognosis is seen in patients with high collagen expression, who also express MITF target genes such as the differentiation markers TRPM1, TYR and TYRP1, as well as CDK4. In summary, we reveal a distinct lineage-specific route of YAP signalling that contributes to the regulation of melanoma pigmentation and uncovers a set of potential biomarkers predictive for poor survival.

Fibroblasts promote the collective invasion of ameloblastoma tumor cells in a 3D coculture model.

Ameloblastoma is a benign tumor of the odontogenic epithelium with several histological subtypes. All subtypes of ameloblastoma contain abundant stroma; the tumor cells invade collectively into the surrounding tissues without losing intratumor cell attachments. However, the molecular mechanisms mediating ameloblastoma invasion remain unclear. Here, we evaluated the functional significance of the interactions between ameloblastoma tumor cells and stromal fibroblasts on collective cellular invasion using a three‐dimensional cultivation method, double‐layered collagen gel hemisphere (DL‐CGH) culture. The AM‐1 plexiform and AM‐3 follicular human ameloblastoma cell lines and HFF‐2 human fibroblasts were labeled with GFP and DsRed, respectively. Collective cellular invasion of ameloblastoma cells was assessed in the presence or absence of fibroblasts. Notably, without fibroblasts, AM‐1 cells formed sharp, plexiform‐like invasive processes, whereas AM‐3 cells formed a series of blunt processes often observed during collective migration. In comparison, under the cocultures with HFF‐2 fibroblasts, AM‐3 cells formed tuft‐like invasive processes and collectively invaded into outer layer more than that observed with AM‐1 cells. Moreover, HFF‐2 fibroblasts localized to the tips of the invasive tumor processes. These findings suggest that tumor‐associated cells assist tumor cell invasion. Microscopic analysis of sectioned three‐dimensional cultures revealed that AM‐3/HFF‐2 hemispheres were histologically similar to follicular ameloblastoma tumor samples. Therefore, our findings suggest that ameloblastoma subtypes exhibit distinct invasion patterns and that fibroblasts promote collective tumor invasion in follicular ameloblastoma.

Abstract 4329: Targetable nodes in fibroblast-supported melanoma cells that show resistance to BRAF inhibitors

Abstract Metastatic melanoma is notorious for the ability to change its phenotype in response to signals from the microenvironment, which might influence how melanoma responds to therapy. We have disclosed an association between fibroblast-induced phenotypic alterations in melanoma and resistance to the mutated BRAF inhibitor vemurafenib (BRAFi). This signifies the need to find other targets than BRAF to eliminate stroma-influenced melanoma cells. To approach this challenge, we performed proteomic analysis and cancer drug sensitivity screening, comparing fibroblast-supported versus non-supported melanoma cells. We showed that the effect of fibroblasts was critically dependent on cell-cell proximity, where melanoma cells get trapped in a fibronectin network, produced by adjacent fibroblasts. In such environment, melanoma cells down-regulate melanocytic programs (MITF-driven), gain mesenchymal features (AXL, PDGFR, fibronectin) and activate stress/inflammatory-response signaling pathways (JNK and STAT3). Altogether, this indicates fibroblast-induced melanoma transition to a de-differentiated, mesenchymal-like, pro-inflammatory phenotype. Melanoma cells with such phenotype were less responsive to BRAF/MAPK inhibitors and a number of other targeted drugs. However, they showed enhanced sensitivity to PI3K/mTOR inhibitors and, particularly, an inhibitor of GSK3b, stimulating Wnt/b-catenin signaling. Further, we employed flow cytometry to measure the levels of Ki67 and pS6 in single melanoma cells upon different conditions/treatments. Such analysis allowed discrimination of cell subpopulations representing a proliferative and a quiescent cellular state, and nicely reflected the influence of the tested drugs in the presence or absence of fibroblasts. We observed a subpopulation of proliferative pS6high/Ki67high melanoma cells, which remained after treatment with BRAFi if fibroblasts were present. This, fibroblast-protected BRAFi-resistant cell subpopulation, could be reduced/eliminated by PI3K or GSK3b inhibitors, verifying PI3K/GSK3 as potential targets in fibroblast-rich tumors. Currently, we are using mass cytometry (CyTOF) to further characterize cell subpopulations with respect to multiple markers related to cell signaling and immune interactions. Preliminary results indicate that not only signaling protein levels, but also levels of immunoregulatory proteins are altered in melanoma cells that get support from the fibroblasts. In conclusion, we demonstrate fibroblast-induced melanoma switching to a mesenchymal-like pro-inflammatory phenotype, which favors melanoma resistance to BRAF inhibitors, but sensitizes to inhibitors of PI3K/mTOR-associated signaling. CyTOF-analysis of complex tumor-stroma cell systems is used to search for additional strategies to target stroma-supported melanoma cells, either at the level of signaling, or immune interactions. Citation Format: Kotryna Seip, Marco V. Haselager, Kjetil Jørgensen, Marco Albrecht, Mads H. Haugen, Eivind Valen Egeland, Philippe Lucarelli, Thomas Sauter, Olav Engebraaten, Gunhild M. Mælandsmo, Lina Prasmickaite. Targetable nodes in fibroblast-supported melanoma cells that show resistance to BRAF inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4329. doi:10.1158/1538-7445.AM2017-4329

702 dsRNA induces ectopic KRT9 expression via WNT/β-catenin-mediated signaling

Double stranded RNA (dsRNA;Poly (I:C)) activates innate immunity against viral infection through its receptor, toll-like receptor 3 (TLR3). Previously, we reported that dsRNA released by wounds also triggers TLR3- to enhance hair regeneration, coincident with activation of WNT/β-catenin signaling. Other groups have shown that WNT/β-catenin signaling is required for the thick epidermis of the palms and soles. Thus, we hypothesized that dsRNA, through WNT/β-catenin, could enhance volar (palmo-plantar) skin features. Using genome-wide analysis, we show that KERATIN 9 (KRT9) is the most uniquely enriched transcript in volar keratinocytes (P<0.05, N=3). KRT9 is activated by volar compared to non-volar fibroblast in vitro co-cultures (P<0.01, N=4); we also find, however, that induction of KRT9 in non-volar keratinocytes alone occurs in the absence of fibroblasts. In both cases, ectopic expression of KRT9 is either activated (P<0.01, N=3) or inhibited (P<0.02, N=3) through the WNT pathway agonist SB216763 or siRNA of β-catenin respectively demonstrating the overlapping paracrine or autocrine requirement of WNT/β-catenin. To begin testing the connection to dsRNA, we find it stimulates the expression of WNT5a and β-catenin (P<0.05, N=3) and induces nuclear localization of β-catenin(P<0.01, N=3). Coincidentally, Poly (I:C) robustly increases KRT9 expression in keratinocytes alone and in co-cultures with volar fibroblasts (P<0.05, N=5). KRT9 expression induced by Poly (I:C) was attenuated by loss of function β-catenin with either knockdown or DKK-1 treatment (P<0.05, N=3-4). Finally to demonstrate the clinical relevance of keratinocyte cell autonomous KRT9 expression apart from volar fibroblasts, we find ectopic expression of KRT9 in the human skin disease Lichen Simplex Chronicus (LSC) coincident with WNT5a expression. Collectively, ours results suggest a model where damage from friction/weight might release dsRNA and augment KRT9 in palms and soles through β-catenin signaling.

Abstract B16: Progastrin activates colon fibroblasts and participates to the dialogue between tumor epithelial cells and stromal fibroblasts in colorectal cancer

Abstract Colorectal cancer (CRC) represents the second leading cause of cancer-related death worldwide. One of the main reasons for this high mortality rate is because CRC develops into invasive lesions with high metastasis potential. The cancer-associated fibroblasts (CAFs) present in the tumour reactive stroma promote epithelial cancer cell motility and aggressiveness. However, whereas the role of tumour-stroma crosstalks in cancer progression is increasingly documented, how “normal” resident fibroblasts are converted into pro-invasive CAFs and how CAFs participate to the epithelial-stroma dialogue remains an open question. Precursor of the hormone gastrin, progastrin, now established as a growth factor for colon cells, is detected in colorectal polyps and tumours while it is absent in healthy colon. Using immunohistochemical and biochemical approaches we found that progastrin induces the expression of fibroblast activation markers (alphaSMA, FAPalpha) in the normal colon mucosa of progastrin-overexpressing mice as well as in the human colon fibroblast cell line CCD18Co. To investigate the role of progastrin-induced fibroblast activation, we compared the migration of colon epithelial cancer cells expressing or not the hormone in presence or absence of fibroblasts in Boyden chamber. Progastrin-expressing epithelial cells have increased migration capacities in presence of fibroblasts while not difference between the two epithelial cell lines was observed in absence of fibroblasts. Moreover, we identified the implication of different chemokines in this process. Thus, here we demonstrate that progastrin, secreted by tumour epithelial cells, contributes to the activation of the fibroblasts present in the cancer-associated stroma and participates to the epithelial-stroma dialogue. Citation Format: Nicolas Fénié, Claudine Bertrand, Aurélien Lacombe, Serge Roche, Corinne Bousquet, Valérie Gouazé-Andersson, Christine Toulas, Elizabeth Moyal, Audrey Ferrand. Progastrin activates colon fibroblasts and participates to the dialogue between tumor epithelial cells and stromal fibroblasts in colorectal cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B16. doi:10.1158/1538-7445.CHTME14-B16

In Vivo Biocompatibility Testing of a Collagen Cell Carrier Seeded with Human Urothelial Cells in Rats

Tissue-engineered, matrix-stabilized autologous urothelium is a new option for urethral reconstruction, particularly for patients for whom other autologous grafts are not available. In vitro engineering of urothelial tissue requires biomaterials as cell carriers that increase the stability of cell-based implants. The aim of this study was to investigate a new highly standardized, industrially manufactured bovine collagen type I-based cell carrier (CCC) for its suitability as a carrier matrix for human urothelial cells (HUC). As an in vivo biocompatibility test the behaviour and degradation of these implants was to be proven in a nude rat model. Expanded HUC from tissue biopsies were phenotypically analysed by immuno¬cyto¬chemistry and seeded onto CCC. For in vivo application, CCC was seeded with PKH26-labelled HUC in high density and constructs were implanted onto the rectus abdominis muscle of nude rats. Integration, cell survival, and degradation of the urothelium-matrix-implants were studied after 1, 2, and 4 weeks. Immunohistological characterization of multilayered urothelium-matrix-constructs was performed for AE1/AE3 and p63 (epithelial phenotype), CK20 (differentiation), and E-Cadherin and ZO-1 (junction formation). Immunocytochemical staining showed urothelial character of the isolated cells and the absence of fibroblasts and muscle cells. In twelve nude rats, urothelium-matrix-implants integrated well into the host tissue where no inflammation was observed. Immuno¬fluorescence analysis confirmed epithelial phenotype, adherence and tight junction formation homogeneous multilayer formation of stratified HUC cultures as well as urothelial phenotype and differentiation in vitro the CCC-matrix additionally revealed promising biocompatibility investigated in a nude rat model. Thus, this study demonstrates excellent suitability of CCC as a matrix for urothelial cells and recommends its use in a large animal model with regard to clinical application.

Differential cytokine response by macrophages to ManLAM of Mycobacterium tuberculosis under various culture conditions (INC7P.419)

Abstract Lipoarabinomannan, a lipoglycan is a major virulence factor for Mycobacterium. Mannosylated form of Lipoarabinomannan (ManLAM) is commanly found in pathogenic Mycobacterium tuberculosis. It has been shown that ManLams inhibit production of TNFα and IL-12 by macrophages under in vitro culture conditions. In this study, we tested cytokine responses of macrophages stimulated with ManLAM under various culture conditions. ManLAM was isolated from M. tuberculosis strain H37Rv (ATCC 25618). Macrophages (J744.1; murine macrophage cell line) were stimulated with ManLAM in three different conditions, either alone or in co-cultures with fibroblasts (3T3; ATCC, CRL1658) in contact-culture and non-contact culture systems. Cytokines were measured using commercially available ELISA kits. In comparison to unstimulated controls, TNFα, IL-6 and IFNγ levels increased significantly in co-culture systems after stimulation with ManLAM while a slight increase occurred for IL-12 in the same cultures. However, we observed no change in TNFα, IL-6 and IL-12 for stimulated macrophages when they were cultured alone in the absence of fibroblasts. These results suggested that macrophage activity to ManLAM changed in the presence of the fibroblast in co-culture systems that might be an in vitro model for granuloma formation in tuberculosis infections.

The use of allodermis prepared from Euro skin bank to prepare autologous tissue engineered skin for clinical use

Over the past two decades a range of 3D models for human skin have been described. Some include native collagen and intrinsic basement membrane proteins and fibroblasts, others are based on xenogeneic collagen or synthetic supports often without fibroblasts. The aim of this study was to look at the influence of media calcium, basement membrane and fibroblasts on the quality of 3D tissue engineered skin produced using human de-epidermized acellular dermis. In this study we deliberately used Euro skin as the source of the donor dermis to examine to what extent this could provide an effective dermal substrate for producing 3D skin for clinical use. Keratinocytes were cultured in the presence and absence of fibroblasts and both with and without basement membrane on decellularized dermis at calcium concentrations ranging from 250μM to 1.6mM over a period of 14 days. Results showed the formation of a well attached epithelium with many of the features of normal skin in the presence of a basement membrane. This was largely independent of the presence of fibroblasts and not greatly influenced by the concentration of calcium in the media. However there was a clear requirement for physiological levels of calcium in the formation of a stratified epithelium in the absence of a basement membrane.

IL1β-mediated Stromal COX-2 signaling mediates proliferation and invasiveness of colonic epithelial cancer cells

COX-2 is a major inflammatory mediator implicated in colorectal inflammation and cancer. However, the exact origin and role of COX-2 on colorectal inflammation and carcinogenesis are still not well defined. Recently, we reported that COX-2 and iNOS signalings interact in colonic CCD18Co fibroblasts. In this article, we investigated whether activation of COX-2 signaling by IL1β in primary colonic fibroblasts obtained from normal and cancer patients play a critical role in regulation of proliferation and invasiveness of human colonic epithelial cancer cells. Our results demonstrated that COX-2 level was significantly higher in cancer associated fibroblasts than that in normal fibroblasts with or without stimulation of IL-1β, a powerful stimulator of COX-2. Using in vitro assays for estimating proliferative and invasive potential, we discovered that the proliferation and invasiveness of the epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts than with normal fibroblasts, with or without stimulation of IL1β. Further analysis indicated that the major COX-2 product, prostaglandin E2, directly enhanced proliferation and invasiveness of the epithelial cancer cells in the absence of fibroblasts. Moreover, a selective COX-2 inhibitor, NS-398, blocked the proliferative and invasive effect of both normal and cancer associate fibroblasts on the epithelial cancer cells, with or without stimulation of IL-1β. Those results indicate that activation of COX-2 signaling in the fibroblasts plays a major role in promoting proliferation and invasiveness of the epithelial cancer cells. In this process, PKC is involved in the activation of COX-2 signaling induced by IL-1β in the fibroblasts.

Fibroblasts as negative regulators of inflammation: implications for the pathogenesis of rheumatoid arthritis?

Backgroundand objectivesFibroblasts possess immune regulatory capacities. They are able to suppress the proliferation of T lymphocytes and to modulate the differentiation of dendritic cells (DC). Being ubiquitously present at the...

Abstract 3104: Stromal fibroblasts promote progression of esophageal squamous cell carcinoma cells both in vitro and in vivo

Abstract Objectives: The purpose of the study was to clarify the role of fibroblasts in the proliferation and growth pattern of esophageal squamous cell carcinoma and to experimentally study interactions between cancer cells and fibroblasts. Methods: We used 22 esophageal squamous cell carcinoma cell lines (mainly, TE series and KYSE series obtained from the RIKEN Cell Bank and the Japanese Collection of Research Bioresources, respectively) and human esophageal fibroblasts that had been established in our laboratory from surgical specimens. Cancer cells were cultured on Matrigel in the presence or absence of fibroblasts in the Matrigels, and their growth patterns were examined. Cell count assays were performed to examine the effects of the fibroblast culture supernatant, growth factors, cytokines, and various inhibitors on cancer cell growth. We also examined the influence of co-injection of fibroblasts on tumor growth in immunodeficient mice. Results: When plated on Matrigel, nearly all cell lines spread and grew in an invasive manner in the presence of fibroblasts, while in the absence of fibroblasts, most cell lines formed round or spheroidal colonies. Cell-count assay showed that in most cancer cells, cell proliferation was stimulated by addition of fibroblast supernatant. In particular, cell proliferation increased by more than 50% in 11 cell lines. Addition of hepatocyte growth factor (HGF) or interleukin-6 elicited a definitive proliferative response in only 2 and 3 cell lines, respectively. Both KYSE-220 and KYSE-50 proliferated in the presence of HGF, but the addition of MET inhibitor (PHA665752) suppressed the proliferation of only the former. In vivo tumor growth was stimulated by co-injection of fibroblasts in 9 of 22 cancer cell lines. Conclusions: Fibroblast had a profound influence on cell growth pattern on Matrigel. In many esophageal cancer cell lines, cell proliferation was promoted by the presence of fibroblasts both in vivo and in vitro. The stimulatory effect of fibroblast could be accounted for by HGF and/or interleukin-6 in some, but not in all, esophageal cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3104. doi:10.1158/1538-7445.AM2011-3104

Fully automated, quantitative, noninvasive assessment of collagen fiber content and organization in thick collagen gels.

Collagen is the most prominent protein of human tissues. Its content and organization define to a large extent the mechanical properties of tissue as well as its function. Methods that have been used traditionally to visualize and analyze collagen are invasive, provide only qualitative or indirect information, and have limited use in studies that aim to understand the dynamic nature of collagen remodeling and its interactions with the surrounding cells and other matrix components. Second harmonic generation (SHG) imaging emerged as a promising noninvasive modality for providing high-resolution images of collagen fibers within thick specimens, such as tissues. In this article, we present a fully automated procedure to acquire quantitative information on the content, orientation, and organization of collagen fibers. We use this procedure to monitor the dynamic remodeling of collagen gels in the absence or presence of fibroblasts over periods of 12 or 14 days. We find that an adaptive thresholding and stretching approach provides great insight to the content of collagen fibers within SHG images without the need for user input. An additional feature-erosion and feature-dilation step is useful for preserving structure and noise removal in images with low signal. To quantitatively assess the orientation of collagen fibers, we extract the orientation index (OI), a parameter based on the power distribution of the spatial-frequency-averaged, two-dimensional Fourier transform of the SHG images. To measure the local organization of the collagen fibers, we access the Hough transform of small tiles of the image and compute the entropy distribution, which represents the probability of finding the direction of fibers along a dominant direction. Using these methods we observed that the presence and number of fibroblasts within the collagen gel significantly affects the remodeling of the collagen matrix. In the absence of fibroblasts, gels contract, especially during the first few days, in a manner that allows the fibers to remain mostly disoriented, as indicated by small OI values. Subtle changes in the local organization of fibers may be taking place as the corresponding entropy values of these gels show a small decrease. The presence of fibroblasts affects the collagen matrix in a manner that is highly dependent on their number. A low density of fibroblasts enhances the rate of initial gel contraction, but ultimately leads to degradation of collagen fibers, which start to organize in localized clumps. This degradation and reorganization is seen within the first days of incubation with fibroblasts at a high density and is followed by de novo collagen fiber deposition by the fibroblasts. These collagen fibers are more highly oriented and organized than the fibers of the original collagen gel. These initial studies demonstrate that SHG imaging in combination with automated image analysis approaches offer a noninvasive and easily implementable method for characterizing important features of the content and organization of collagen in tissuelike specimens. Therefore, these studies could offer important insights for improving tissue engineering and disease diagnostic efforts.