It is a well established phenomenon that the epithelial lining of the urinary bladder (urothelium) when implanted into skeletal muscle induces ectopic ossification. However, despite numerous observations, this reaction is poorly understood. This research further studied this reaction by - (a) demonstrating the reaction in a suitable small animal model; (b) attempting to induce the reaction by implanting urothelial cells purified by cell culture techniques; and (c) comparing the bone forming reaction induced by implanted urothelium to the reaction induced by implanting Bone Marrow Stem Cells (BMSC's) and Osteophyte Stem Cells (OSC's). By demonstrating newly formed bone after the implantation of guinea pig urothelium into the skeletal muscle of a Severe Combined Immuno-Deficient Mouse (SCID-Mouse) this research demonstrated that a suitable small animal model had been established. This is despite inherent difficulties (particularly bacterial contamination) associated with establishing a primary cell culture of guinea pig urothelial cells. Additionally, the intramuscular ectopic osteoinductive potential of human BMSC's (hBMSC's) in the SCID-mouse has also been demonstrated. Confirming that the injection of cultured cells in suspension is an adequate intramuscular delivery technique, this research demonstrates that hBMSC's induce ectopic ossification by non-immunological means. This research has demonstrated a number of differences between urothelium induced ectopic ossification and ectopic ossification induced by BMSC's, suggesting they are two separate processes. This is important because the chemotaxis and subsequent osteogenic differentiation of BMSC's has previously been one of the more popular postulated mechanisms of urothelium induced ectopic ossification. Finally, this research has demonstrated the ectopic osteoinductive potential of stem cells isolated from the marrow of human osteophytes (human Osteophyte Stem Cells, hOSC's). This observation has not been previously reported, and will hopefully provide a valuable contribution to a body of knowledge that has important ramifications in both the treatment of osteoarthritis, and the use of BMSC's in tissue engineering.

Low back pain is a common cause of disability and work inability, often associated with intervertebral disc degeneration. The current understanding of disc degeneration is limited and none of the available treatments is entirely effective. The work described herein investigates potential strategies for the biological herapeutic restoration of disc degeneration. Firstly, an in vitro study to investigate the effects of BMP-7 on human discal cellular viability was performed. Cultured cells were treated with TNF-a or H202 to induce apoptosis, resulting in the down regulation of extracellular matrix proteins, decreased cell viability, morphological changes and activation of caspase-3; however, addition of BMP-7 alone prevented the observed effects, demonstrating the ability of BMP-7 to prevent apoptosis of human disc cells in vitro. Secondly, the differentiation potential of stem cells towards disc-like cells was studied. Rodent mesenchymal stem cells (rMSCs) were cultured alone or co-cultured with rat disc tissue. Differentiation potential of rMSCs was evaluated by mRNA and protein expression, cellular function and morphological studies. The co-culture conditions led to the expression of chondrocytic markers in rMSCs, whereas rMSCs cultured alone did not express the chondrocytic markers. Cellular contact between the co-cultured rMSCs and the discal tissue were observed. This study demonstrated that rMSCs can differentiate into functional disc-like cells in a tissue influenced co-culture environment. Finally, the survival and differentiation of CD34+ or CD34?? bone marrow (hBM) cells, in an intra-discal xenogeneic transplantation rat model was investigated. Human CD34+ or CD34?? cells were isolated, fluorescent-labelled and injected into rat coccygeal discs. The survival of transplanted cells was confirmed by fluorescent positive cells as well as a human nuclear specific marker. Interestingly, CD34?? cells survived until day 42 in the injected discs, and differentiated into cells ex:pressing a chondrocytic phenotype. In contrast, CD34+ cells could not be detected by day 21. This data suggests that transplanted hBM CD34?? cells, in contrast to CD34+ cells, were able to survive and differentiate within the intervertebral disc. Together, the results of these studies can both encourage and contribute to the basis of potential biological therapies in the restoration of intervertebral disc degeneration.

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 143 pages. Includes Vita. Includes bibliographical references.

Submitted by FERNANDA HERRERA DA COSTA (ferherreracosta@gmail.com) on 2018-05-24T14:02:59Z No. of bitstreams: 1 dissertação final.pdf: 14796170 bytes, checksum: b56cc2f9884a2482b189d839e1ebdcca (MD5) / Approved for entry into archive by Silvana Alvarez null (silvana@ict.unesp.br) on 2018-06-06T19:33:04Z (GMT) No. of bitstreams: 1 godoi_fhc_me_sjc.pdf: 14796170 bytes, checksum: b56cc2f9884a2482b189d839e1ebdcca (MD5) / Made available in DSpace on 2018-06-06T19:33:04Z (GMT). No. of bitstreams: 1 godoi_fhc_me_sjc.pdf: 14796170 bytes, checksum: b56cc2f9884a2482b189d839e1ebdcca (MD5) Previous issue date: 2018-03-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A sobrevida de pacientes submetidos a tratamentos para câncer de cabeça e pescoço está aumentando e a busca por reabilitação é necessária para promover qualidade de vida. O objetivo deste estudo é avaliar o perfil da expressão gênica de transcritos relacionados na osteogênese e osteoclastogênese em uma cultura primária de células mesenquimais de fêmures de ratos submetidos a radioterapia e colocação de implantes de titânio. Setenta e dois ratos receberam implantes de titânio em ambos fêmures. Os animais foram divididos em quatro grupos: 1) Grupo controle (C): cirurgia de colocação dos implantes; 2) Implante + irradiação imediata (IrI): irurgia de implantes e seguido de irradiação imediata; 3) Implante + irradiação tardia (IrT): cirurgia de implantes e irradiação após 4 semanas; 4) Irradiação prévia + implantes (IrP): irradiação e após 4 semanas cirurgia de implantes. A dose de irradiação foi de 30 Gy fracionadas em duas sessões. As eutanásias nos períodos de 3, 14 e 49 dias após o tratamento. A células cultivadas sofreram diferenciação em osteoblastos. A expressão gênica dos genes Fosfatase alcalina (Alp), Colágeno 1 (Col-1), Integrina !1 (Itg ! ), Osteocalcina (Bglap), Osteopontina (Osp), Osteonectina (Osn), Sialoproteína Óssea (Bsp), Fator de crescimento transformador (Tgf- ! ), Osterix (Osx),Fator relacionado ao Runt (Runx2), Fator estimulador de colônias de macrófagos (M-csf), Interleucina -6 (Il-6) Apolipoproteína E (Apo-E), Prostaglandina E2 (PgE2), foram avaliados por qRT-PCR e os resultados validados por ELISA. A expressão mais alta de Alp foi encontrada no grupo IrP (p=0.0001) e foi subexpressa nos grupos IrI e IrT (p<0.0001 e p=0.0041 respectivamente). Resultados similares foram encontrados nos transcritos de Itg !, On, Bsp, Osx e Runx2. mRNA do Tgf- ! foi hiperexpresso em todos os grupos principalmente aos 49 dias. Depois de 49 dias, os níveis de proteína da Bglap e Il-6 foram correlacionados com a expressão do mRNA. A radioterapia imediata altera a atividade de diferenciaçãO das células mesenquimais dos fêmures de ratos. / Prognosis of patient submitted to head and neck cancer treatment has increased and the oral rehabilitation becomes necessary to improve their life quality. The aim of this study was to evaluate the gene expression profile of transcripts related to osteogenesis and osteoclastogenesis in primary culture of mesenchymal cells from rat femurs submitted to radiotherapy and installation of pure titanium implant. Seventy two rats received titanium implants in both femurs. The animals were divided in four groups: 1- Control (C) implant surgery; 2- Implant + immediate irradiation after 24 hours (IrI); 3- Implant + late irradiation after 4 weeks (IrL); 4- Implant + Previous irradiation 4 weeks before surgery (IrP) irradiation. The irradiation dose was 30Gy fractioned in two. The animals were euthanized in day 3, 14 and 49 after surgery. The mesenchymal cells from femurs were extracted and cultivated. The differentiation into osteoblastic cells was verified by calcification nodules formation. The gene expression of Alkaline Phosphatase (Alp), Collagen 1 (Col 1), Integrin β1 (ItgB1), Osteocalcin (Bglap), Osteopontin (Osp), Osteonectin (Osn), Bone Sialoprotein (Bsp), Transforming Growth Factor β-type (Tgf-β), Osterix (Osx), Runt-related transcription factor 2 (Runx2), Macrophage Colony- Stimulating Factor (M–csf), Interleukin-6 (Il-6), Apolipoprotein E (ApoE) and Prostaglandin E2 (PgE2) were evaluated by qRT-PCR and the results were validated by ELISA test. Higher mRNA of Alp expression was found in IrP group (p=0.0001) and it was downregulated in IrI and IrT groups (p<0.0001 and p=0.0041, respectively). Similar results were found for transcript levels of ItgB1, Osn, Bsp, Osx and Runx2. mRNA of Tgf-β was overexpressed in all groups mainly in 49 days. After 49 days, Osn and Bsp transcripts were downregulated in the 3 groups evaluated. The Bglap and IL-6 protein level was correlated to their mRNA expression. The radiotherapy alters immediately the differentiation and activity of mesenchymal cells from rat femurs; however these cells seem to recover becoming suitable for receiving implants. / FAPESP: 2016/20103-7

Articular cartilage plays a vital role in load-bearing joints, providing an almost frictionless surface to articulating bones. However, the avascular nature and low cell density of the tissue means that following injury, there is limited potential for regeneration and repair. With the ageing population, the prevalence and economic burden associated with osteoarthritis (OA) is increasing rapidly, but as of yet there are no fully effective ways to treat the condition. Research into novel therapies has therefore become a popular avenue of investigation, and human mesenchymal stem/stromal cells (hMSCs) have been highlighted as particularly promising targets. However, current, methods for inducing the chondrogenic differentiation of hMSCs, which typically employ the use of transforming growth factor beta 1 or 3 (TGFβ1/3), result in the production of hypertrophic rather than hyaline tissue, hampering translational progress. Growth differentiation factor 5 (GDF5) belongs to the TGFβ superfamily of proteins and is vital for skeletal formation, however its use in cartilage tissue engineering (TE) strategies has been somewhat neglected. Here we demonstrate that GDF5 significantly increases aggrecan gene expression (a marker of articular cartilage), without affecting collagen type X expression (a marker of chondrocyte hypertrophy), in chondrocyte pellet cultures derived from hMSCs, making it a promising target for the formation of permanent articular cartilage. The therapeutic application of growth factors is, at present, limited due to their expense, susceptibility to proteolytic degradation, and rapid clearance, leading to large quantities being required to get anywhere near the desired outcome. The highly sulfated glycosaminoglycan (GAG), heparin, is already extensively used in the clinic as an anticoagulant, and is also able to bind and potentiate the activity of a wide range of growth factors. As such, researchers are now using it to enhance stem cell expansion/ differentiation protocols, as well as to improve the delivery/ activity of growth factors in TE strategies. Here, we identify GDF5 as a novel heparin/heparan sulfate (HS)-binding protein, and show that endogenous HS proteoglycans (HSPGs) are vital for localizing GDF5 to the cell surface, but are not required for its signalling activity. Importantly, we report that clinically relevant doses of heparin (≥ 10 nM), but not equivalent concentrations of HS, inhibit GDF5’s biological activity, in both hMSC-derived chondrocyte pellet cultures, and in the skeletal cell line ATDC5. We demonstrate that these inhibitory effects are due to heparin (but not HS) inhibiting both GDF5 binding to endogenous HSPGs and GDF5-induced induction of Smad 1/5/8 signalling. This study may therefore explain the variable (and disappointing) results seen with heparin-loaded biomaterials for skeletal TE, and the adverse skeletal effects, such as osteoporosis, that have been reported in the clinic following long-term heparin treatment. Together, our results caution the use of heparin in the clinic and in TE applications, and prompt the transition to using more specific GAGs (e.g. HS derivatives or synthetics), with better-defined structures and fewer off-target effects, if optimal therapy is to be achieved. In the case of GDF5, we have used a variety of developed techniques to begin uncovering important structural and functional information regarding the HS-GDF5 interaction, which are hoped to ultimately pave the way towards achieving this aim. Although further analysis is necessary, our data indicate that relatively long HS sequences are required for binding, and that both ionic and non-ionic interactions play a role in the interaction. In addition we suggest that low- rather than high-affinity HS variants may be key to potentiating the activity of this growth factor.

La perte de l’expression de la sous-unité β4 des canaux sodiques dépendants du voltage NaV dans les tumeurs mammaires est associée à un grade cancéreux élevé et au développement des métastases. L’extinction de son expression dans les cellules MDA-MB-231 augmente de plus de deux fois leur invasivité. Au cours de cette thèse, nous avons montré que la sous-expression de β4 favorise la transition mésenchymato-amoeboïde et augmente l’invasion cancéreuse indépendante de NaV. Cette transition se caractérise par l’acquisition d’une morphologie plus arrondie, par la présence de blebs à la surface cellulaire et par une augmentation de l’activité RhoA-GTPase. Cette transition est inhibée par la surexpression du domaine intracellulaire C-terminal de la sousunité β4. L’expression de β4 peut être augmentée par un apport en acide docosahexaènoïque (22:6n-3), qui augmente l’activité du promoteur de son gène SCN4B. Le DHA augmente également l’expression de β4 en modulant l’expression des récepteurs nucléaires PPAR, sensibles aux lipides. / The loss of voltage gated sodium channel NaVβ4 subunit expression in breast cancer biopsies is associated with high grade tumors and metastatic development. The inhibition of β4 expression in MDA-MB-231 breast cancer cells enhanced their invasiveness by two fold. During this thesis, we have shown that β4 underexpression promotes mesenchymal-amoeboid transition and increases NaV-independent invasion. This transition is characterized by rounded morphology, the presence of blebs at the cell surface and an increased RhoAGTPase activity. This transition is inhibited by β4 C-terminal intracellular domain overexpression. Expression of β4 can be enhanced by a DHA supplementation that increases the encoding SCN4B promoter activity. DHA also increases β4 expression through the modulation of PPARs lipid-sensitive nuclear receptors expression.

The ovarian surface epithelium (OSE) is a monolayer of cells surrounding the ovary that is ruptured during ovulation. After ovulation the wound is repaired, however this process, and the mechanisms to maintain OSE homeostasis after the wound is repaired are poorly understood. We have shown the mouse OSE (mOSE) contains a stem cell population that is expanded by Transforming Growth Factor Beta 1 (TGFB1), a factor present in follicular fluid. These data suggest that components in the follicular fluid such as TGFB1 may promote wound repair and OSE homeostasis through maintenance of the OSE stem cell population. Additionally, TGFB1 may promote wound repair through induction of an epithelial-to-mesenchymal transition (EMT) and activation of pro-survival pathways, as seen in other tissues. To elucidate the mechanism for TGFB1-mediated ovulatory wound repair, mOSE cells were treated with TGFB1, which induced an EMT seen with increased Snai1 expression and cell migration. Snai1 overexpression also increased cell migration and sphere formation (a stem cell characteristic). RNA sequencing results suggest this is at least in part through elevated collagen deposition in SNAI1 overexpressing cells. A TGFB signalling targets array identified Cox2 induction following TGFB1 treatment. Constitutive Cox2 expression did not promote an EMT, but enhanced sphere formation and cell survival. Finally, TGFB1 treatment decreased Brca1 expression, which when deleted from mOSE cells also increased sphere formation. RNA sequencing results suggest that Brca1 deletion promotes stemness through activation of the stem cell genes Ly6a and Lgr5. RNA sequencing was also used to compare mOSE cells cultured as monolayers and as spheroids, with and without TGFB1. These results validate our findings that TGFB1 promotes an EMT partially through Snail induction and the upregulation of Cox2. mOSE cells cultured as spheroids acquire a mesenchymal transcriptional profile that is further enhanced with TGFB1 treatment. These data suggest that TGFB1 may promote ovulatory wound repair and maintain OSE homeostasis through the induction of an EMT, maintenance of the stem cell population and activation of a pro-survival pathway. Interestingly, mOSE spheroids also decrease Brca1 expression and upregulate cancer associated genes such as Pax8 and Greb1. The induction of survival pathways, while simultaneously increasing stemness and repressing Brca1 could render cells more susceptible to transformation. This work provides novel insights as to why ovulation is the primary non-hereditary risk factor for ovarian cancer.

Le cancer gastrique (CG) est une maladie multifactorielle, fréquemment associée à l’infection chronique par des souches CagA+ d’Helicobacter pylori. La transition épithélio-mésenchymateuse (EMT) est un processus réversible dans lequel une cellule épithéliale polarisée acquiert un phénotype mésenchymateux. L’EMT est à l’émergence de cellules souches cancéreuses (CSC) qui expriment CD44 et présentent une activité ALDH élevée. L’infection des cellules épithéliales gastriques humaines (CEGs) par CagA+ H. pylori induit des cellules CD44+ avec des propriétés des CSCs via une EMT. La voie Hippo est composée par les kinases MST et LATS, et leurs cibles, les YAP1 et TAZ. Suite à la phosphorylation, YAP1 et TAZ sont inhibés. YAP1 et TAZ activés lient les facteurs TEAD pour promouvoir la croissance cellulaire et l’inhibition de l’apoptose.Notre premier objectif était de rechercher si H. pylori change l’état d’activation de la voie Hippo et l'effet sur l’EMT et les CSC in vitro et in vivo. Le deuxième but est la caractérisation du rôle de YAP1/TEAD dans les propriétés de CSCs gastriques in vitro et les conséquences de son inhibition dans la croissance tumorale in vivo.Pour étudier la régulation de la voie Hippo pendant l’infection par H. pylori, LATS2, YAP1 et CD44 ont été évalués dans la muqueuse gastrique de sujets non-infectés et infectés par H. pylori, qui ont été augmentés avec l’infection et leur surexpression a été associée avec la gastrite et la métaplasie intestinale. Dans les CEGs l’expression de gènes de la voie Hippo a été altérée par l’infection. La régulation de la voie Hippo par H. pylori a une cinétique diphasique et dépendante de CagA. Dans l’infection précoce, H. pylori déclenche l’activité transcriptionelle de YAP1. Cette période d’inactivité de la voie Hippo est suivi de son activation progressive, soutenue par l’accumulation de LATS2 et la phosphorylation inhibitrice de YAP1. La répression de LATS2 avec siRNAs a accéléré l’acquisition du phénotype mésenchymateux après l’infection, l’augmentation de marqueurs de l’EMT (Zeb1 et Snail1), et la diminution des miR-200 épithéliaux. Les CSC induites par H. pylori ont été potentialisées par l’inhibition de LATS2, ce qui suggère que LATS2 limite l’EMT et le phénotype de CSC acquis pendant l’infection. L’inhibition de LATS2 ou YAP1 diminue l’expression de ces deux protéines, révélant ainsi une boucle de régulation positive. Dans des coupes de tissu de CG, l’expression de LATS2 et YAP1 est hétérogène et positivement corrélée, fait qui a été confirmé dans 38 CEGs de la CCLE. L’expression LATS2 est fortement corrélée à celle de CTGF et CYR61, ce qui suggère que LATS2 peut aussi être un gène cible de YAP1/TEAD.La verteporfine (VP) est capable d’interrompre l’interaction YAP1/TEAD, et donc d’inhiber son activité transcriptionelle. In vitro, utilisant CEGs et des cellules de tumeurs de patients amplifiées chez la souris (patient-derived xenograft PDX), le traitement à la VP a diminué la croissance cellulaire, l’expression de gènes cible de YAP1/TAZ/TEAD, l’activité du rapporteur TEAD-luciférase et la capacité de formation de sphères. L’activité de la VP a été testée in vivo par injection péri-tumorale dans un modèle de greffe sous-cutanés des CEGs MKN45 et MKN74 et le PDX GC10 chez la souris NSG. La croissance tumorale a été diminuée. Le poids des tumeurs, l’analyse par IHC (CD44, ALDH, Ki67) et la capacité de formation de sphères des CSCs résiduelles ont été diminuées. Ces résultats montrent une activité inhibitrice de la VP sur les CSCs gastriques in vitro et in vivo.Ce travail montre pour la première fois que l’axe LATS2/YAP1/TEAD est précocement activé pendant l’infection chronique avec H. pylori et que celui-ci contrôle l’EMT et les propriétés de CSC. Le ciblage de la voie Hippo a été montré comme étant efficace dans la prévention de la croissance tumorale, mettant en évidence le potentiel de son inhibition dans le traitement du cancer gastrique. / Gastric cancer (GC) is a multifactorial disease, most frequently associated to chronic infection with CagA-positive Helicobacter pylori strains. Epithelial-to-mesenchymal transition (EMT) is reversible process in which polarized epithelial cells acquire a mesenchymal phenotype. EMT is at the origin of cancer stem cells (CSC). In GC, CSCs express CD44 and high aldehyde-dehydrogenase (ALDH) activity. Infection with H. pylori of human gastric cancer cell lines (hGECs) in vitro induces the emergence of a population of CD44+ cells with CSC-properties through an EMT process in a CagA-dependent manner. The Hippo pathway is composed by the kinases MST and LATS, and their phosphorylation targets,YAP1 and TAZ. Upon phosphorylation by LATS, YAP1 and TAZ are inhibited. Active YAP1 and TAZ bind to TEAD transcription factors to promote the expression of genes that regulate cell growth and apoptosis.The first aim of this work was to investigate whether H. pylori affects the activation state of the Hippo pathway, and its effect on the EMT process and the CSCs. Second, we intended to characterize the role of YAP1/TEAD in gastric CSC properties in vitro and the consequences of its pharmacological inhibition on tumor growth in vivo.To study the Hippo pathway regulation during infection, LATS2, YAP1 and CD44 were evaluated in gastric mucosae of non-infected or H. pylori-infected patients. They were upregulated in infected mucosae and were associated to pathology. Hippo pathway regulation by H. pylori infection has biphasic kinetics and is CagA-dependent. Early in infection, H. pylori transiently triggered YAP1 expression and co-transcriptional activity, along with LATS2. This period of Hippo pathway inactivity is followed by a progressive activation, sustained by LATS2 accumulation and inhibitory YAP1Ser127-phosphorylation. LATS2 siRNA-mediated repression accelerated the acquisition of the EMT-phenotype upon infection, the up-regulation of EMT-markers ZEB1 and Snail1, and the decrease of the epithelial miR-200. H. pylori-induced CD44 upregulation, invasion and sphere-forming capacity were further enhanced upon LATS2 knockdown, suggesting that LATS2 restricts the EMT and CSC-like phenotype in hGECs upon H. pylori infection. Inhibition of either LATS2 or YAP1 reduced the expression of both proteins, revealing a positive feedback loop. In tissue sections of GC, LATS2 and YAP1 were heterogeneous and co-expressed. The positive correlation between LATS2 and YAP1 was confirmed in the 38 hGECs of the CCLE. The expression of CTGF and CYR61 was also strongly correlated to LATS2, suggesting that LATS2 could also be a YAP1/TEAD target gene.hGECs of the CCLE. The expression of CTGF and CYR61 was also strongly correlated to LATS2, suggesting that LATS2 could also be a YAP1/TEAD target gene.Verteporfin (VP) disrupts the YAP1/TEAD interaction inhibiting its transcriptional activity. In vitro, using hGECs and cells from patient derived primary tumor xenogratfs (PDXs), we showed that treatment with VP decreased cell growth, expression of YAP1/TAZ/TEAD target genes, TEAD-luciferase reporter activity and sphere-forming capacity. The activity of VP was tested in vivo, by peritumoral injection in a model of subcutaneous graft of hGECs (MKN45 and MKN74) and PDX (GC10) in NGS mice. Tumor growth was followed and a decrease was observed. Tumor weight measurement, IHC analysis (CD44, ALDH and Ki67), and CSCs were decreased in treated tumors. These results show the CSC-inhibitory activity of VP both in vitro and in vivo.We showed for the first time that the LATS2/YAP1/TEAD axis is early activated during the carcinogenesis process induced by chronic H. pylori infection and controls the subsequent EMT and CSC-like features. Targeting the Hippo pathway efficiently prevented tumor growth in a PDX model, highlighting the potential of its inhibition to be implemented in gastric cancer therapy.

Colorectal cancer (CRC) is the fourth most common cause of cancer related deaths in the UK with the prognosis dependent on the degree of tumour invasion and presence of metastasis at diagnosis. An important step in the invasion and metastasis of solid tumours is the loss of cell-cell junctions and the acquirement of a more motile mesenchymal phenotype which is facilitated by the epithelial-mesenchymal transition (EMT). The presence of EMT is linked with a more aggressive, invasive tumour and subsequent poor prognosis. In addition to roles in motility and invasion, EMT can induce a cancer stem cell phenotype in a subset of tumour cells. Cancer stem cells (CSCs) are a subpopulation of cells capable of self-renewal and maintaining a cellular population whilst displaying increased therapeutic resistance. Induction of EMT and CSCs can be regulated by common signalling pathways with expression of EMT transcription factors inducing CSCs expression. Understanding the signalling pathways regulating EMT and CSC formation in cancer is important for preventing of metastasis and combating therapeutic resistance. Aspirin’s role in cancer prevention has been established for a number of years with aspirin treatment reducing the incidence of CRC. Recently, evidence has emerged suggesting aspirin treatment may have post-diagnosis benefits and increase survival rates of CRC patients. A potential mechanism for the post-diagnosis benefit of aspirin is the inhibition of EMT and CSC formation which both facilitate tumour progression and metastasis. Aspirin has been demonstrated to suppress the migratory and invasive capacity of lung cancer cell lines by inhibiting EMT. Whilst aspirin has been shown to inhibit platelet-induced EMT in CRC, the direct effects of aspirin on EMT in CRC cell lines has not been established. I hypothesis that aspirin inhibits cell migration, invasion and EMT in CRC which results in a reduction in the CSC population and contributes to the clinical benefit of post-diagnosis aspirin. Using CRC cell lines, I have demonstrated that aspirin treatment inhibits cell migration, invasion, motility and promotes an epithelial phenotype. These results have been confirmed in human organoids and mouse intestinal adenoma in vivo models. Aspirin also promotes a budding phenotype in Apc deficient organoids and reduces expression of stem cell markers in both mouse and human tissue. Aspirin inhibits the mTOR and Wnt signalling pathways in vivo which have the ability to regulate EMT and CSCs although signalling dependency has not been determined. Regardless, aspirin is decreasing the cancer stem cell population and promoting a non-invasive epithelial phenotype which may explain some of the previously described post-diagnosis benefits.

Magnetic hyperthermia (MH) is an anti-cancer treatment which exploits the heat produced by tumour-targeted magnetic nanoparticles (MNPs) subjected to an alternating magnetic field (AMF). A problem limiting the clinical use of MH, however, is the inability to adequately localise the MNPs at the tumour site. A cellular approach using mesenchymal stem cells (MSCs) as carriers has been proposed as these cells are believed to home to sites of tissue injury and tumour growth, however problems with MNPs uptake and toxicity retard progress and need to be overcome. The aim of this project was to find an alternative approach in MH treatment, creating engineered human MSCs able to biosynthesise MNPs. To achieve this goal, MSCs were transfected with either, or both, M. magneticum AMB-1 mms6 and mmsF genes. M. magneticum AMB-1 is a genus of magnetotactic bacteria, containing magnetosomes, which are lipidic organelles containing single crystals of magnetite. M. magneticum-AMB1 mms6 and mmsF genes are important for final crystal morphology and are known to play a role in crystal synthesis and growth respectively. The originality of this study was in using mms6 and mmsF genes, which were codon-optimized for mammalian expression, alone or in combination, for transfection of human MSCs, which have known tumour homing capacity. The transfected MNPs-bearing MSCs, able to migrate into the tumour tissue, were subjected to AMF in MH experiments in an attempt to induce cancer cell death. mms6 and mmsF gene expression, following transfection, was investigated in the human osteosarcoma cell line MG63 by reverse transcription polymerase chain reaction (RT-PCR). The cellular ultrastructure of transfected MG63 cells was investigated by transmission electron microscopy (TEM), revealing the presence of nanoparticles. The magnetism of transfected MG63 cells was proved by superconducting quantum interference device (SQUID) and supported by in vitro MH experiments. Then, human MSCs were transfected with mms6 and mmsF genes, alone or in combination. The effect of transfection experiments and MNPs synthesis on MSCs markers of stemness, cell proliferation and differentiation ability were investigated. The MTB genes expression in human MSCs was assessed by RT-PCR and cell magnetism was confirmed by SQUID, in vitro MH experiments and by magnetic force microscopy (MFM). Then, in vitro studies of MH were undertaken to establish whether mms6 transfected MSCs expressing MNPs supported a MH effect when exposed to an AMF. Cells were initially exposed to an AMF of 565.3 kHz frequency in monolayers and in 3D arrangements and cell death/viability was assessed. Subsequently, the effect of the same AMF on 3D models of mixed populations of mms6-expressing MSCs and cancer cells was assessed. The results indicate that viability of MNPs-expressing MSCs and adjacent cancer cells is reduced following AMF exposure. In vivo studies of MH were undertaken following intracardiac injection of mms6-expressing MSCs in tumour-bearing mice (epidermoid carcinoma). The expression of mms6-expressing MSCs inside mice organs was confirmed by RT-PCR, fluorescence microscopy and immunohistochemistry. The effect of the application of an AMF of 565.3 kHz on mice tumours was studied with different techniques (tumour size and volume measurement, multiphoton microscopy, haematoxylin and eosin staining, and activated Caspase 3 expression), to understand if MNPs created inside mms6- expressing MSCs, following AMF exposure, could lead to cancer cell death. Results indicate that mice tolerate the treatment well, however no appreciable tumour reduction or necrosis was evident. Overall the results suggest that mms6 transfection alone confers the highest magnetisation to MSCs compared to mmsF alone or mms6+mmsF co-transfected, and that mms6 expression in human MSCs does not have an adverse effect on important cell functions. mms6-expressing MSCs, when exposed to an AMF, show reduced viability and enhanced cell cytotoxicity in vitro. When co-cultured with cancer cells in 3D models in vitro, mms6-expressing MSCs are able to reduce viability of adjacent cancer cells confirming the potential applicability of mms6- expressing MSCs for MH treatment. In vivo proof of concept experiments show that mms6-expressing MSCs can locate to the tumour tissue, and mms6-expressing intracardiac injected MSCs mice exposed to AMF tolerate the treatment well. However, the number of mms6-expressing MSCs able to localize to the tumour tissue in this experiment was too low to give an appreciable tumour reduction, so more experiments are needed to enhance the experimental protocol. A number of improvements are required to progress this novel technique towards clinical application. Gene transfection and MNPs production need to be optimised, the best frequency for MH needs to be established and MSCs delivery to the tumour has to be significantly increased to allow concentration of MNPs. The study has helped to increase our knowledge on the creation of magnetic human MSCs to potentially use these cells in MH cancer treatment.