In Canada, colorectal cancer is the second leading cause of cancer death for both men and women. There are many different factors that contribute to the progression and spread of the disease. However, increasing evidence now suggests that the tumor microenvironment plays a paramount role in these processes. CD26 is a multifunctional, cell-surface glycoprotein that has intrinsic enzyme activity, binds adenosine deaminase and interacts with the extracellular matrix. Through its various functions it serves to constrain cancer progression. For example, it is known to cleave CXCL12, the ligand for CXCR4. The CXCL12:CXCR4 axis is normally involved in cancer metastasis by promoting cancer cell migration, invasion and proliferation. Down-regulation of CD26 is observed in certain cancers - this has been shown in vitro to occur in response to certain soluble mediators. The first part of this study looked at the effects of glucose and its metabolic product lactate on CD26 expression in colorectal carcinoma cells. Our study showed that CD26 expression is lower in cancer cells that are grown in low-glucose, high-lactate conditions, which replicates the situation within a tumor. The second part of this study examined the effect of adenosine, a purine nucleoside, on colorectal carcinoma cells and supportive stromal cells - cancer-associated HS675.T fibroblasts (CAFs) and Met-5a mesothelial cells. Adenosine increased the proliferation of CAFs and increased CXCL12 mRNA in both stromal cell lines. It also increased MMP-13 mRNA in stromal cells as well as colorectal cancer cells, suggesting that adenosine may promote progression and metastasis through various mechanisms. The last section focused on the ability of cellular products and 3-dimensional tissue topology to coordinate and affect the behaviour of the different cell populations. Here we show that secretory products from colorectal cancer cells promote CAF proliferation but inhibit mesothelial cell proliferation, and are also able to modulate MMP-13 expression. Finally, certain responses are enhanced in multicellular spheroids. In conclusion, the tumor microenvironment represents a major consideration in the treatment of solid tumors. Our data suggest that various soluble mediators, such as adenosine, may have therapeutic implications in cancer treatment and might represent novel targets for future research.

FES protein-tyrosine kinase (PTK) activation downstream of the KIT receptor in mast cells (MC) promotes cell polarization and migration towards the KIT ligand Stem cell factor (SCF). A variety of tumours secrete SCF to promote MC recruitment and release of mediators that enhance tumour vascularization and growth. This study investigates whether FES promotes MC migration via regulation of microtubules (MTs), and if FES is required for MC recruitment to the tumour microenvironment. MT binding assays showed that FES has at least two MT binding sites, which likely contribute to the partial co-localization of FES with MTs in polarized bone marrow-derived mast cells (BMMCs). Live cell imaging revealed a significant defect in chemotaxis of FES-deficient BMMCs towards SCF embedded within an agarose drop, which correlated with less MT organization compared to control cells. To extend these results to a tumour model, mouse mammary carcinoma AC2M2 cells were engrafted under the skin and into the mammary fat pads of immune compromised control (nu/nu) or FES-deficient (nu/nu:fes-/-) mice. A drastic reduction in tumour-associated MCs was observed in FES-deficient mice compared to control in both mammary and skin tissue sections. This correlated with a trend towards reduced tumour volumes in FES-deficient mice. These results implicate FES signaling downstream of KIT, in promoting MT reorganization during cell polarization and for chemotaxis of MCs towards tumour-derived SCF. Thus, FES is a potential therapeutic target to limit recruitment of stromal mast cells or macrophages to solid tumours that enhance tumour progression. / Thesis (Master, Biochemistry) -- Queen's University, 2011-09-14 11:49:32.871

Diffuse large B-cell lymphoma (DLBCL), which is the most common type of lymphoma, is characterised by its aggressiveness and poor outcome without adequate treatment and also for its biological and clinical heterogeneity. It is therefore highly desirable to gain a more profound understanding of the underlying biology of the disease, as well as predictive factors for the guidance of treatment. The studies presented here attempt to gain an overall grasp on DLBCL, from the epidemiological level down to the genomic level. The tumour microenvironment consists of both tumour cells and normal infiltrating cells in a delicate interplay. By assessing the number of infiltrating mast cells (MCs) in the microenvironment, a correlation between low numbers of MCs and poorer prognosis of DLBCL was found. However, malignant cells are not only affected by environmental conditions but also by intrinsic factors, such as small non-coding microRNAs. A low expression level of microRNA-129 was found to correlate with poor survival of DLBCL and the finding remained significant even for rituximab-treated patients. An even smaller intracellular genomic unit is one single nucleotide. The single nucleotide polymorphism 309 (SNP309) is a T to G change in the promotor region of MDM2, a regulatory protein in the p53 pathway, which results in increased transcription of MDM2 and thus decreased levels of p53. It was found that homozygous T allele patients had longer overall survival, as well as disease-specific survival and disease-free survival. However, treatment with rituximab eliminated the predictive value of the SNP309 polymorphism. In the last project presented in this thesis we used epidemiological methods to analyse all DLBCL cases diagnosed 2000-2013 in Sweden. Here it was possible to categorically show that higher age is an adverse prognostic factor, and most importantly, this starts from a young age. In conclusion, within this thesis I have applied different laboratory and analysis techniques to examine DLBCL biology in relation to the clinic. I have identified potential new prognostic markers, contributed to an enhanced understanding of DLBCL biology and described epidemiological data from one of the largest DLBCL cohorts ever presented. All of these aspects provide important information for a deeper understanding of the disease DLBCL.

Antigens play a critical role in the development of chronic lymphocytic leukemia (CLL) by binding to and stimulating leukemic precursor cells at some point during CLL ontogeny. Nevertheless, much remains unknown and further studies are necessary before an accurate model of antigen-drive can be ascertained. In this context, intraclonal diversification (ID) analysis of immunoglobulin (IG) genes could shed light on whether antigen involvement is restricted to the malignant transformation phase or if the triggering antigen(s) continuously stimulates the CLL clone. Hence, in Paper I we conducted a large-scale analysis of 71 CLL cases and revealed that 28/71 cases carried intraclonally diversified IGHV-IGHD-IGHJ genes. Although most cases showed no or low levels of ID, intense ID was evident within all subset #4 (IGHV4-34/IGKV2-30) cases. Subsequent analysis, in Paper II, of the clonotypic light chains revealed that the outstanding exception again related to subset #4. In such cases, the expressed IGKV2-30 gene was affected by targeted ID, analogous to their partner IGHV4-34 gene. Whilst these results convincingly argued for the role of antigen(s) in the development and evolution of CLL subset #4, this analysis was limited to depicting what was occurring at a single time-point and could not provide insight into the temporal dynamics of the CLL clones. Thus, in Paper III we conducted a longitudinal study of 8 subset #4 cases which enabled us to establish a hierarchical pattern of subclonal evolution. The observed ‘stepwise’ accumulation of mutations strongly supports a role for antigen selection in the pathogenesis of CLL subset #4. In Paper IV we reported a subset of IgG-switched CLL patients with coexisting trisomies of 12 and 19, and propose that the emergence of trisomy 18 in such cases represents a clonal evolution event suggestive of selection due to a clonal advantage. Paper V focused on the IGHV3-21 gene, an adverse prognostic factor in CLL. Since ~60% of IGHV3-21-expressing cases carry stereotyped B cell receptors, recognition of a common antigenic epitope, perhaps of pathogenic significance, is envisaged. Therefore, we investigated IGHV3-21 gene frequency within a Swedish population-based cohort and assessed the impact of stereotypy on clinical outcome. Taken collectively, this thesis provides molecular and genetic evidence for the role of antigen in CLL pathogenesis by convincingly demonstrating that clonal evolution, at least for certain subsets of CLL, is functionally driven rather than a consequence of clonal expansion promoted by nonspecific stimuli.

Notch signaling is often and aberrantly activated by hypoxia during tumor progression; however, the exact pathological role of hypoxia-induced Notch signaling in tumor metastasis is as yet poorly understood. In the first part of this study, we aimed to define the mechanism of Notch ligand activation by hypoxia in both primary tumor and bone stromal cells in the metastatic niche and to clarify their roles in tumor progression. We have analyzed the expression profiles of various Notch liagnds in 779 breast cancer patients in GEO database and found that the expression of Jagged2 among all five ligands is most significantly correlated with the overall- and metastasis-free survival of breast cancer patients. The results of our immunohistochemical (IHC) analysis for Jagged2 in 61 clinical samples also revealed that both Jagged2 and Notch signaling were strongly up-regulated at the hypoxic invasive front. Activation of Jagged2 by hypoxia in tumor cells induced EMT and also promoted cell survival in vitro. Notably, a ã-secretase inhibitor significantly blocked Notch-mediated invasion and survival under hypoxia by promoting expression of E-cadherin and inhibiting Akt phosphorylation. Importantly, Jagged2 was also found to be up-regulated in bone marrow stroma under hypoxia and promoted the growth of cancer stem-like cells by activating their Notch signaling. Therefore, hypoxia-induced Jagged2 activation in both tumor invasive front and normal bone stroma plays a critical role in tumor progression and metastasis, and Jagged2 is considered to be a valuable prognostic marker and may serve as a novel therapeutic target for metastatic breast cancer. In the second part of this study, the role of Notch signaling in brain metastasis was investigated. Metastatic diseases are responsible for the majority of the deaths in breast cancer patients and the brain is one of the most common metastatic sites. The metastatic tumor in the brain profoundly affects the cognitive and sensory functions as well as morbidity of patients, and the one year survival rate among these patients remains less than 20%. However, the pathological mechanism of brain metastasis is as yet poorly understood. In this report, we found that metastatic breast tumor cells in the brain highly expressed IL-1â which can "activate" astrocytes. This activation significantly augmented the expression of JAG1 in the reactive astrocytes, which in turn activated Notch signaling pathway of cancer stem-like cells (CSCs) upon direct interaction. We also found that the activated Notch signaling in CSCs up-regulated Sox2 followed by promoting self-renewal of CSCs. Furthermore, we have shown that the blood-brain barrier permeable Notch inhibitor, Compound E, can significantly suppress the brain metastasis growth in our animal model. These results represent a novel paradigm for the understanding of how metastatic breast CSCs re-establish their niche for their self-renewal in a totally different microenvironment, which opens a new avenue to identify a novel and specific target for the brain metastatic disease

Orientador: Noeme Sousa Rocha / Resumo: O tumor venéreo transmissível (TVT) é um câncer transmissível, que propaga-se naturalmente entre os cães, pela transferência alogênica de células tumorais, principalmente, durante o coito. Sabe-se que o microambiente tumoral influencia diretamente no comportamento tumoral, além de interagir com o sistema imune do hospedeiro. Face aos escassos estudos relacionados aos eventos celulares envolvidos no microambiente tumoral, o presente estudo teve por objetivo quantificar a expressão de genes relacionados com esse microambiente, tanto em células do TVT in vivo como in vitro, correlacionando-o com a resposta clínica dos animais tratados com vincristina. Para tal, foram incluídos neste estudo 18 cães atendidos no Hospital Veterinário da FMVZ-UNESP, Câmpus de Botucatu, portadores de TVT de ocorrência natural. As amostras tumorais foram coletadas antes do tratamento quimioterápico, cultivadas e submetidas à análise citogenética e quantificação através de RT-qPCR. Observou-se amostras tumorais com reduzido número cromossomos (59) em relação ao cão (78) e o subtipo celular prevalente foi o plasmocitoide, embora células com padrão linfocitoide também tenham sido observadas. Quando comparamos as expressões gênicas, o gene TP53 se mostrou elevado na maioria dos casos, indicando possível alteração nesse gene, enquanto BCL-2, BCL-xL, BAX e RASSF1 apresentaram-se variáveis, dependendo do animal analisado, fato que pode ser devido a alterações genéticas e/ou epigenéticas no TVT, além da possi... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

O microambiente tumoral é composto por células, como fibroblastos, células do sistema imune, células endoteliais e pericitos, envoltas por uma matriz extracelular, além de possuir fatores solúveis que participam da comunicação celular. Nas últimas décadas, têm-se entendido cada vez melhor seu papel na iniciação e progressão dos tumores. É de fundamental importância, portanto, entender a biologia dos seus componentes e como podem agir em favor do desenvolvimento tumoral. Diversos trabalhos demonstram que há uma associação entre a presença dos pericitos nos vasos tumorais com a agressividade e prognóstico de alguns tipos de câncer. Uma vez ativadas, além do papel estrutural, essas células modulam as atividades das células endoteliais durante a formação de novos vasos, além de adquirirem propriedades como proliferação e migração. Neste contexto, os pericitos passam a secretar fatores importantes na comunicação célula-a-célula e liberam enzimas moduladoras na matriz extracelular. A lisil oxidase (LOX) é uma das principais enzimas que atuam sobre a matriz extracelular. Já está bem descrito que, quando superexpressa em células tumorais, a LOX pode alterar a migração e invasão dessas células, promovendo a geração de metástases. Entretanto, pouco se sabe a respeito da atuação dessa enzima sobre os demais componentes celulares do estroma tumoral, como os pericitos. Sendo assim, o presente trabalho teve como objetivo principal verificar se enzima LOX é relevante para a ativação de propriedades dos pericitos que possam contribuir para suas funções pró-tumorigênicas, como migração, proliferação e formação de vasos. Os resultados foram gerados avaliando essas atividades dos pericitos após pré-tratamento de 24 horas com β-aminopropionitrile (βAPN), um inibidor irreversível da LOX. Foram utilizadas duas linhagens de pericitos derivados de tecido normal (adiposo e muscular) e duas linhagens de pericitos provenientes de tecido tumores do sistema nervoso central (neuroblastoma e ependimoma). Este composto foi capaz de diminuir a capacidade de migração das células de todas as linhagens testadas e, de maneira geral, tornou o processo de formação de estruturas tubulares in vitro menos eficiente. Entretanto, não foram observadas alterações na proliferação celular. Os dados indicam, portanto, que a enzima LOX pode ser importante para a ativação dos pericitos e, possivelmente, influenciem no seu comportamento no microambiente tumoral / The tumor microenvironment is composed of non-cancer cells, such as fibroblasts, immune cells, endothelial cells and pericytes, surrounded by an extracellular matrix, in addition to soluble factors involved in cellular crosstalk. In the last decades, it has been better understood its role in the initiation and progression of tumors. It is critical, therefore, to understand the biology of its components and how they can act in favor of tumor development. Several studies show an association between the presence of pericytes in tumor vessels with aggressiveness and prognosis of some cancers. Once activated, these cells modulate the activities of endothelial cells during the new vessels formation, and acquire properties as proliferation and migration. In this context, pericytes triggers the secretion of important factors in cell-to-cell communication and release modulating enzymes of extracellular matrix. The lysyl oxidase (LOX) is one of the main enzymes that act on the extracelular matrix. It is well described that when overexpressed in tumor cells, LOX can alter the migration and invasion of these cells, promoting the generation of metastases. However, little is known about the role of this enzyme over other cellular components of the tumor stroma, such as pericytes. Therefore, the aim of this study was to verify whether LOX enzyme is relevant to the activation of properties of the pericytes that could contribute to its pro-tumorigenic functions such as migration, proliferation and vessel formation. All the results were generated by evaluation of the activities of these pericytes after 24 hours pretreatment with β-aminopropionitrile (βAPN), an irreversible inhibitor of LOX. This study used two cell lines of pericytes derived from normal tissue (fat and muscle) and two isolated from tissue of the central nervous system. The βAPN was able to reduce the migration of cells of all tested cell lines and, in general, alter the tubular formation in vitro. However, changes in cell proliferation weren′t observed. The data showed, that the LOX family may be important for the activation of pericytes and possibly influence on their behavior in the tumor microenvironment

Orientador: Jose Andres Yunes / Tese (doutorado) - Universidade Estadual de Campinas. Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T09:06:10Z (GMT). No. of bitstreams: 1 Vasconcellos_JairaFerreirade_D.pdf: 12382146 bytes, checksum: 9d05fdf79968e31e12ded32312675286 (MD5) Previous issue date: 2010 / Resumo: A leucemia linfóide aguda (LLA) é a neoplasia mais freqüente na infância. As interações dos blastos da LLA com as células do estroma da medula óssea (MO) têm um impacto positivo na sobrevivência das células e resistência a quimioterapia. A LLA estimula as células do estroma da MO que reciprocamente promovem a sobrevivência da leucemia. Para identificar moléculas envolvidas na interação leucemia-microambiente foi realizada análise do perfil de expressão gênica de células mesenquimais (MSC) da MO estimuladas com células primárias da LLA. O estímulo da LLA nas MSC ativou várias quimiocinas próinflamatórias, incluindo CCL2 e IL-8. Os níveis plasmáticos de CCL2 e IL-8 em crianças com LLA ao diagnóstico foram significativamente maiores do que em controles normais. A maioria das amostras de LLA primária expressou transcritos dos receptores de CCL2 e IL-8. Ensaios funcionais in vitro demonstraram que a LLA não é afetada pela adição de CCL2, IL- 8 ou anticorpos neutralizantes. Porém ambas as quimiocinas demonstraram estimular a sobrevivência das MSC em meio sem soro e aumentar sua proliferação em meio com quantidades limitadas de soro. Para explorar o efeito da IL-8 no microambiente da MO leucêmica foi sintetizado um antagonista do receptor CXCR2 da IL-8, denominado SB225002 (N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea). O SB225002 demonstrou efeito deletério contra as linhagens da LLA (Nalm6, REH, Jurkat, CEM e Molt4). Mas nem todas as linhagens da LLA sensíveis ao SB225002 expressaram o receptor CXCR2, sugerindo que seu mecanismo de ação ocorreria através de receptor alternativo. Recentemente foi descrito que o SB225002 também se liga a outros receptores acoplados a proteína G, dentre eles os receptores da histamina e dos canabinóides. Ambos foram testados in vitro e o receptor CNR2 dos canabinóides demonstra desempenhar função no mecanismo de ação do SB225002. Além disso, para identificar moléculas envolvidas na resposta celular ao SB225002 foi realizada a análise do perfil de expressão gênica de células Jurkat tratadas com SB225002. Eventos celulares de resposta inicial ao SB225002 incluíram (i) ativação de phospho-p44/42 ERK e (ii) ativação de GLIPR1 que demonstrou mediar a indução de morte do SB225002. Em conclusão, este trabalho indica a importância das quimiocinas CCL2 e IL-8 no microambiente da LLA, e demonstra o potencial do SB225002 como agente antileucêmico. / Abstract: The interactions of Acute Lymphoblastic Leukemia (ALL) blasts with bone marrow (BM) stromal cells have a positive impact on leukemia cell survival and resistance to chemotherapy. ALL stimulates BM stromal cells, which reciprocally promote leukemia cell survival. To identify molecules critically involved in leukemia-microenvironment crosstalk, we performed gene expression profiling analyses of primary BM mesenchymal stem cells (BMMSC) following stimulation by primary ALL cells. Leukemia stimulation of BMMSC up regulated the expression of several inflammatory chemokines, including CCL2 and IL-8. Secretion of these molecules was confirmed by ELISA assays of in vitro co-culture experiments and in BM plasma samples from pediatric ALL patients. Most primary ALL samples were found to express mRNA for CCL2 and IL-8 receptors. In vitro functional studies revealed that primary ALL cells co-cultured with BMMSC were not affected by addition of CCL2, IL-8 or neutralizing antibodies to these chemokines. On the other hand, both chemokines were found to enhance BMMSC survival in serum-free medium and to increase their proliferation in serum-starved conditions. To further explore the effect of IL-8 in the ALL-BM microenvironment the CXCR2 -IL-8 receptor-antagonist SB225002 ( N - ( 2 - hydroxyl - 4 - nitrophenyl ) - N' - ( 2 - bromophenyl ) urea) was synthesized. SB225002 had a deleterious effect against ALL cell lines (Nalm6, REH, Jurkat, CEM, and Molt4). Suprisingly, not all the ALL cells lines that were sensitive to SB225002 expressed CXCR2 receptor. This find suggested that the SB225002's mechanism of action occurred through a different receptor. SB225002 was recently described to also bind histamine and cannabinoid receptors that were investigated in ALL and the CNR2 cannabinoid receptor demonstrated to play a role in SB225002 mechanism of action. To identify molecules involved in the cellular effects promoted by SB225002, gene expression profiling analyses was performed of Jurkat cells treated with SB225002. Early cellular effects enhanced by SB225002 included (i) activation of phospho-p44/42 ERK and (ii) up regulation of GLIPR1 that shown to mediate SB225002-induced apoptosis. In conclusion, this work support a significant role for the chemokines CCL2 and IL-8 in the ALL-BM microenvironment, and demonstrate SB225002's therapeutic potential. / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Tissue engineering is an interdisciplinary field which aims to create substitutes of damaged tissues by combining cells with biomaterials. Cells are extremely sensitive to their microenvironment and so the cell response to biomaterials can be regulated by different extrinsic stimuli and alterations of biomaterial properties. Successful implant integration into the tissue can therefore be promoted by appropriate surface roughness, chemical composition, adhesion ligand density, as well as the availability of growth factors. This thesis mainly focuses on the development of orthopedic replacements and the improvement of the currently used blood vessel prostheses. Through the study of cell-biomaterial interactions, it was demonstrated that superimposed topography with features ranging from the nano to micro scale promotes cell spreading, proliferation, and the metabolic activity of osteoblast-like cells. Moreover, when comparing the chemical composition of biomaterials for orthopedic implants, higher osteoblast densities were observed on composites with 5-15 vol. % of calcium phosphate nanoparticles, while concentrations of 25 vol. % did not support cell proliferation. Cell viability, however, was not affected. In vivo, a more intensive formation of new bone tissue, was found on samples containing...

Oncolytic viruses (OVs) have been engineered or selected for cancer cell-specific infection; however, we have found that following intravenous administration of vesicular stomatitis virus (VSV), tumour cell killing rapidly extends far beyond the initial sites of infection. This Bystander Effect is due to the virus’ ability to specifically target tumour vasculature through tumour-specific infection of tumour endothelium and the induction of an inflammatory response resulting in tumour-restricted coagulation, acute vascular disruption, apoptosis and necrosis of the tumour core. VSV-infected tumours, reconstructed in three-dimensions from serial histological sections, revealed that the majority of the tumour mass lacks significant blood flow in contrast to uninfected tumours, which exhibit relatively uniform perfusion. VSV infection rapidly induced intravascular coagulation within 6 hours of intravenous administration. The induction of coagulation was dependent on neutrophils and could be prevented with inhibitors of the coagulation pathway. Normal vasculature was not infected by VSV and no increase in coagulation was observed. Vascular collapse was also observed with the oncolytic poxvirus, JX-594, in patients and preclinical models. Biopsies from patients enrolled in a dose escalation trial for JX-594 were immunoreactive for vaccinia antigens and transgene products in high dose cohorts. Tumour-associated vessels from patients treated with JX-594 were infected with JX-594 and expressed virally encoded transgenes. A decrease in blood flow was also observed 5 days post infection. Several viruses, VSV, JX-594, vvDD, Maraba, and Sindbis, were able to rapidly induce widespread bystander cell death in a subset of mouse models. Tumours responded to OV therapy in three ways, and the type of response was determined by two factors - susceptibility to infection and the heterogeneity of the tumour microenvironment. Heterogeneity correlated with E-cadherin expression. Among tumours that supported viral replication, cancers with low E-cadherin expression were susceptible to vascular collapse. E-cadherin positive tumours were susceptible to infection and direct cell killing but resistant to vascular disruption or bystander cell death. If poorly-differentiated tumours were resistant to infection, no acute cell killing was observed. These histological subtypes provide a potential framework for the rational selection of patients, the integration of combination therapies and the creation of designer viruses to improve the success of OV therapy.