Heterogeneous cancer-associated fibroblast population potentiates neuroendocrine differentiation and castrate resistance in a CD105-dependent manner.

Heterogeneous prostatic carcinoma-associated fibroblasts (CAF) contribute to tumor progression and resistance to androgen signaling deprivation therapy (ADT). CAF subjected to extended passaging, compared to low passage CAF, were found to lose tumor expansion potential and heterogeneity. Cell surface endoglin (CD105), known to be expressed on proliferative endothelia and mesenchymal stem cells, was diminished in high passage CAF. RNA-sequencing revealed SFRP1 to be distinctly expressed by tumor-inductive CAF, which was further demonstrated to occur in a CD105-dependent manner. Moreover, ADT resulted in further expansion of the CD105+ fibroblastic population and downstream SFRP1 in 3-dimensional cultures and patient-derived xenograft tissues. In patients, CD105+ fibroblasts were found to circumscribe epithelia with neuroendocrine differentiation. CAF-derived SFRP1, driven by CD105 signaling, was necessary and sufficient to induce prostate cancer neuroendocrine differentiation in a paracrine manner. A partially humanized CD105 neutralizing antibody, TRC105, inhibited fibroblastic SFRP1 expression and epithelial neuroendocrine differentiation. In a novel synthetic lethality paradigm, we found that simultaneously targeting the epithelia and its microenvironment with ADT and TRC105, respectively, reduced castrate-resistant tumor progression, in a model where either ADT or TRC105 alone had little effect

Human Galectins Induce Conversion of Dermal Fibroblasts into Myofibroblasts and Production of Extracellular Matrix: Potential Application in Tissue Engineering and Wound Repair

Members of the galectin family of endogenous lectins are potent adhesion/growth-regulatory effectors. Their multi-functionality opens possibilities for their use in bioapplications. We studied whether human galectins induce the conversion of human dermal fibroblasts into myofibroblasts (MFBs) and the production of a bioactive extracellular matrix scaffold is suitable for cell culture. Testing a panel of galectins of all three subgroups, including natural and engineered variants, we detected activity for the proto-type galectin-1 and galectin-7, the chimera-type galectin-3 and the tandem-repeat-type galectin-4. The activity of galectin-1 required the integrity of the carbohydrate recognition domain. It was independent of the presence of TGF-beta 1, but it yielded an additive effect. The resulting MFBs, relevant, for example, for tumor progression, generated a matrix scaffold rich in fibronectin and galectin-1 that supported keratinocyte culture without feeder cells. Of note, keratinocytes cultured on this substratum presented a stem-like cell phenotype with small size and keratin-19 expression. In vivo in rats, galectin-1 had a positive effect on skin wound closure 21 days after surgery. In conclusion, we describe the differential potential of certain human galectins to induce the conversion of dermal fibroblasts into MFBs and the generation of a bioactive cell culture substratum. Copyright (C) 2011 S. Karger AG, Base

Human somatic cells in regenerative medicine : In vitro characterization of mesenchymal stem cells and chondrocytes

Tissue engineering is a new treatment strategy available to patients with injuries and diseases. The goal is to rebuild or replace damaged or dead tissue. The means is stem cells or differentiated organ specific cells, with or without scaffolds to ensure that the new tissue acquires the right shape. The present thesis is based on research performed within cell/stem cell biology, with the aim to improve treatment modalities in regenerative medicine. In order to ensure the best results from tissue engineering treatment strategies, the cells used need to be characterized in detail, and in vitro cell expansion protocols need to be optimalized. In his thesis, cand. scient Aboulghassem Shahdadfar has studied three cell populations with considerable potential in protocols in regenerative medicine. He identified mesenchymal stem cells (MSC) in adipose tissue, and defined their phenotype and gene expression. He showed that these cells could differentiate to several different lineages, and characterized the changes in gene expression induced in these cells by in vitro cell culture. Another population of MSC is found in the bone marrow. Using these cells, Shahdadfar showed that serum from the bone marrow donor could be used for in vitro cell expansion in stead of fetal calf serum, thus avoiding the risk of transfer of zoonoses and immune-stimulating xenogeneic proteins. The cells thus obtained seemed also to be more genetically stable. Chondrocytes obtained from biopsies of articular cartilage has for some time been used for treatment of focal lesions of the hyaline cartilage of the knee. However, in the course of in vitro culture these cells lose the ability to produce the right constituents of hyaline extracellular matrix. Shahdadfar in this thesis presents an improved cell culture method for chondrocytes, which ensures that the cells maintain the ability to produce the right extracellular matrix. Cells cultured in this way are already being used in patients

Investigating the Expression and Function of CCN2 in Articular Cartilage

The pericellular matrix (PCM) is a distinct zone of matrix that surrounds individual chondrocytes throughout articular cartilage. Although not much is known about its function, our group has shown that it has a role in mechanotransduction by controlling the bioavailability of perlecan-bound FGF-2. The aim of this project was to determine the other structural components of the PCM, and to search for other heparin binding growth factors which might be sequestered in the matrix. By confocal microscopy I confirmed that the PCM was rich in type VI collagen and perlecan, although devoid of type II collagen and aggrecan. Isolation of individual chondrons (the chondrocyte together with its PCM) was performed by partial digestion of the matrix using dispase and collagenase, and proteomic analysis using mass spectrometry was performed to identify new proteins. As this method was only partially successful I looked for the presence of a known heparin binding growth factor, connective tissue growth factor (CCN2), in chondron preparations. CCN2, which is an abundant secreted protein of articular cartilage, was present in both the chondron preparation by western blot, and was visualised in the PCM of porcine and human articular cartilage by confocal microscopy. CCN2 is not commercially available so His-tagged recombinant protein was stably expressed and purified using nickel affinity chromatography. Biological activity of the purified protein was investigated in a number of established assays. No biological activity was demonstrated when purified CCN2 was used alone on murine mesenchymal stem cells, but was evident when assayed in combination with low dose TGF-β. The effect of exogenous CCN2 on fibroblasts was limited by the significant release of endogenous CCN2. High constitutive expression of CCN2 in articular cartilage may limit the effects seen by exogenous CCN2. However, the results presented in this thesis support the role of CCN2 as a modulator of TGF beta signalling, and suggest that, through potentiating TGF beta it may regulate matrix turnover in articular cartilage

Differential expression of secreted factors SOSTDC1 and ADAMTS8 cause pro-fibrotic changes in linear morphoea fibroblasts

This is the peer reviewed version of the following article: Badshah, I. I., et al. "Differential expression of secreted factors SOSTDC1 and ADAMTS8 cause pro-fibrotic changes in linear morphoea fibroblasts." British Journal of Dermatology 0(ja)., which has been published in final form at https://doi.org/10.1111/bjd.17352. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsFunding: RO, IB and SB are funded by the Great Ormond Street Children's Charity. This research was supported by the NIHR Great Ormond Street Hospital Biomedical Research Centr

Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells

Background: CCN2/CTGF is known to be involved in tooth germ development and periodontal tissue remodeling, as well as in mesenchymal tissue development and regeneration. In this present study, we investigated the roles of CCN2/CTGF in the proliferation and differentiation of periodontal ligament cells (murine periodontal ligament-derived cell line: MPL) in vitro. Results: In cell cultures of MPL, the mRNA expression of the CCN2/CTGF gene was stronger in sparse cultures than in confluent ones and was significantly enhanced by TGF-β. The addition of Recombinant CCN2/CTGF (rCCN2) to MPL cultures stimulated DNA synthesis and cell growth in a dose-dependent manner. Moreover, rCCN2 addition also enhanced the mRNA expression of alkaline phosphatase (ALPase), type I collagen, and periostin, the latter of which is considered to be a specific marker of the periosteum and periodontium; whereas it showed little effect on the mRNA expression of typical osteoblastic markers, e.g., osteopontin and osteocalcin. Finally, rCCN2/CTGF also stimulated ALPase activity and collagen synthesis. Conclusion: These results taken together suggest important roles of CCN2/CTGF in the development and regeneration of periodontal tissue including the periodontal ligament.</p

An In Vitro 3D Model to Evaluate Behaviour of Breast Cancer Cells and Response to Treatment

The field of 3D culture models of disease has started to move towards systems that aim to recapitulate the complexity of human tissues. However, despite recent improvements, current 3D systems remain overly simplistic, lacking the biophysical characteristics and diverse structures found in most organs. In this project, the cellular behaviour of breast cancer and their responsiveness to chemotherapeutic agents were evaluated under different 3D cell culture conditions. MDA-MB231 and SKBR3 cells were prepared as spheroids using ultra-low attachment plates and as 'artificial cancer masses' (ACM) by embedding cells in a dense collagen type-I. The ACMs were maintained under flow (150 μL/min) and flow/pressure (550 μL/min, ~19 mmHg) conditions. A significant reduction in cell viability was observed when cancer cells were grown as ACM compared to 2D culture. Cell viability also declined significantly when ACMs were maintained in flow/pressure condition compared to static condition. Similarly, an increase in the expression levels of markers of EMT was observed when cells were cultured as ACM. However, compared to static 3D incorporation of flow and pressure was associated with decreased expression levels of vimentin, HIF1-α, whilst MMP14 expression increased and snail remained unchanged. HER2 levels were increased in SKBR3 when the cells were cultured under flow/pressure (1.5 fold) compared to static condition. Overall, cells cultured as ACMs exhibited reduced responsiveness to doxorubicin compared to those grown in the conventional 2D culture. A decrease sensitivity was also observed in 3D/flow/pressure and 3D/flow compared to 3D/static condition. The results obtained in this study show that cancer cell behaviour and their response to therapeutic agents are affected by different microenvironments. Therefore, a new generation of 3D in vitro models need to be developed as pre-clinical drug testing platforms

Development of Novel Strategies for Musculoskeletal Tissue Engineering

The drastic rise in the world’s population coupled to an ever increasing aging population poses a considerable challenge to the orthopaedic community to maintain healthy activity levels. The field of Tissue Engineering and Regenerative Medicine aims to tackle these challenges by implementing more biomimetic strategies to improve upon current treatments. The success of new therapeutic developments in musculoskeletal tissue engineering relies on our ability to study and understand the complex biological interactions between cells, materials, and native tissues so that we may subsequently guide neotissue formation. This thesis is focused on the development of novel, welldefined, and reproducible in-vitro tissue culture models to explore, characterise, and control cellular behaviour and differentiation for osteochondral regeneration. In particular, these models utilised combinations of polymeric biomaterials, differentiated osteoblasts, human periosteal stem cells, and physico-chemical cell signalling cues. In a commercial venture with PolyNovo Ltd (Melbourne, Australia), a novel two-component injectable polymer platform was synthesized and evaluated for uses as a biomaterial construct in orthopaedic applications. The second aspect of this thesis focuses on the harvest, isolation, expansion, and extensive characterisation of human periosteal cells in-vitro. The periosteum is a bi-layered membrane that covers the outside of cortical bone and has been recently identified as a potential stem cell source; with the ability to form osteogenic, chondrogenic, adipogenic, and myogenic tissue types. To detail the heterogeneous cellular features and behaviours of human periosteal cells in-vitro, cells were isolated from surgical explants, expanding in monolayer in the absence of differentiation supplements, and characterised for changes in morphology, growth rate, cell-cycle, gene expression, and phenotype. Additionally, enrichment techniques were designed to preferentially isolate distinct progenitor cell types identified in periosteal cell cultures. Most interestingly, a novel cell-sorting platform utilising droplet microfluidic approaches, was developed and evaluated for its ability to identify and separate periosteal progenitor cells. In the third part of this thesis, a 3-dimensional agarose culture model was created to control and monitor lineage specific human periosteal cell differentiation in various biomechanical and biochemical environments. The work presented herein further demonstrates the potential of human periosteal cells for osteochondral repair and more importantly provides critical information regarding human periosteal cell expansion, phenotype, and differentiation

Modeling Progressive Fibrosis with Pluripotent Stem Cells Identifies an Anti-fibrotic Small Molecule.

Progressive organ fibrosis accounts for one-third of all deaths worldwide, yet preclinical models that mimic the complex, progressive nature of the disease are lacking, and hence, there are no curative therapies. Progressive fibrosis across organs shares common cellular and molecular pathways involving chronic injury, inflammation, and aberrant repair resulting in deposition of extracellular matrix, organ remodeling, and ultimately organ failure. We describe the generation and characterization of an in&nbsp;vitro progressive fibrosis model that uses cell types derived from induced pluripotent stem cells. Our model produces endogenous activated transforming growth factor β (TGF-β) and contains activated fibroblastic aggregates that progressively increase in size and stiffness with activation of known fibrotic molecular and cellular changes. We used this model as a phenotypic drug discovery platform for modulators of fibrosis. We validated this platform by identifying a compound that promotes resolution of fibrosis in in&nbsp;vivo and ex&nbsp;vivo models of ocular and lung fibrosis