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Photodynamic therapy (PDT) is a new form of cancer treatment that uses the localized delivery of light and a photosensitizing drug, which is selectively retained in tumor tissue, to cause photochemically induced cell death. Although PDT mediated by the sensitizer Photofrin (Ph-PDT) is currently in Phase III trials for a number of human cancers, the exact mechanism(s) involved in PDT induced cytotoxicity is not fully understood. Also, Photofrin has a number of drawbacks including extended cutaneous photosensitization and low absorption in the red region of the spectrum. This has lead to the search for improved sensitizers. In vitro, tumor cells resistant to PDT have been developed from PDT sensitive cell lines to examine the mechanism(s) of PDT action. In this work, the sensitivity of RIF-1 murine fibrosarcoma cells and RIF-1 derived Ph-PDT resistant RIF-8A cells was examined following several damaging agents including PDT mediated by the novel Ruthenium phthalocyanine photosensitizer JM2929 (JM2929-PDT), UV, gamma-radiation, and hyperthermia. Gamma-radiation sensitivity of two other RIF-1 derived Ph-PDT resistant variants, CPR-C1 and RIF-P16CL8, was also examined. RIF-8A cells showed cross resistance to UV but increased sensitivity to gamma-rays compared to RIF-1 cells. RIF-1 and RIF-8A cells showed similar sensitivity to JM2929-PDT and hyperthermia. It is possible that Ph-PDT induces a "UV -like" component of damage and/or there is some overlap in the pathways for the repair of UV and Ph-PDT induced damage, but not JM2929-PDT, hyperthermia, and ionizing radiation damage in RIF-1 and RIF-8A cells. A cross resistance to gamma-rays was observed for CPR-C1 but not RIF-P16CL8 cells. Since Ph-PDT resistant CPR-C1 cells, but not RIF-8A cells or RIF-P16CL8 cells, show a cross resistance to gamma radiation, these results suggest that the cellular changes required for RIF-8A, RIF-P16CL8, and CPR-C1 cells to become resistant to Ph-PDT are different. Survival of RIF-1 and RIF-8A cells following gamma-rays in the presence of either Photofrin or JM2929 was also examined. Results suggest sensitization of RIF-1 cells, but not RIF-8A cells, to gamma-radiation in the presence of Photofrin. Gamma-radiation in the presence of JM2929 had no sensitizing effects on the survival of RIF-1 and RIF-8A cells. DNA repair of a UV-damaged reporter gene was also examined in untreated as well as Ph-PDT, JM2929-PDT, UV, cisplatin, and hyperthermia pretreated RIF-1 and RIF-8A cells. Results suggest an increased repair of UV damaged DNA in untreated RIF-1 cells compared to untreated RIF-8A cells. Ph-PDT, JM2929-PDT, and UV pretreatments resulted in an increased reactivation of a UV damaged reporter gene in RIF-1 cells compared to RIF-8A cells. Enhanced reactivation of a UV damaged reporter gene was not observed in either RIF-1 or RIF-8A cells following cisplatin or hyperthermia pretreatment. Enhanced expression of an undamaged reporter gene was greater in RIF-8A cells compared to RIF-1 cells following Ph-PDT pretreatment, but similar to RIF-1 cells following pretreatment with all other agents. These results suggest that the relation between survival, DNA repair of an actively transcribed gene, and transcriptional enhancement of an actively transcribed gene, varies in RIF-1 and RIF-8A cells depending on the damaging agent used. However, decreased reactivation of a UV damaged reporter gene in RIF-8A cells may be related to Ph-PDT and UV resistance seen in RIF-8A cells. / Thesis / Master of Science (MSc)

Tumor-distal immune suppression promotes tumor growth by preventing the recruitment of leukocytes to the tumor-proximal microenvironment. Tumor necrosis factor (TNF)-a is both secreted by and expressed on the cell-surface (mTNF-a) of macrophages. When stimulated with LPS, tumor-burdened host (TBH) macrophages secrete more TNF-a than normal host (NH) macrophages. In this study, I showed that mTNF-a is elevated both in freshly isolated and stimulated TBH macrophages. Additionally, I analyzed the expression of Fas and FasL on freshly isolated and LPS-stimulated macrophages and found no differences between TBH and NH macrophages. Fas and Fas ligand (FasL) cell-surface expression was analyzed on NH and TBH T-cells. While no difference was observed in freshly isolated cells, cell-surface expression of both proteins remained higher in TBH T-cells than NH T-cells after mitogenic stimulation. Fas and FasL analysis was also extended to the MethKDE fibrosarcoma and I found that these tumor cells express high levels of FasL. Because past observations show increased TNF-a mRNA expression in TBH macrophages relative to NH macrophages, I hypothesized that NF-kB activation may be increased as well. NF-kB is a transcription factor whose activation is required for TNF-a transcription. I observed increased NF-kB activation in both splenic and peritoneal TBH macrophages. Interestingly, electrophoretic mobility shift analysis (EMSA) suggests that different species of NF-kB were found in each distinct population of macrophages. Together, these data demonstrate that cell-surface tumoricidal molecules and NF-kB are dysregulated in the tumor-burdened host. / Master of Science

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Assim como tecidos normais, tumores possuem uma demanda de nutrientes e oxigênio, suprida através da vasculatura a eles associada que resulta do processo de angiogênese. Fatores pró-angiogênicos são capazes de atrair monócitos, os quais se diferenciam em macrófagos associados a tumores (TAMs). TAMs comumente apresentam fenótipo M2, cujas características são consideradas pró-tumorais, como a promoção da angiogênese e a degradação de matriz extracelular. Estudos indicam que galectina-3 (gal-3), uma proteína pleiotrópica que se liga a ?-galactosídeos, participa do controle da angiogênese e da infiltração de macrófagos M2 na massa tumoral, mas pouco se sabe sobre os mecanismos envolvidos. No presente estudo, utilizamos um modelo de sarcoma induzido por carcinógeno em camundongos selvagens (WT) e knockout para gal-3 (Gal- 3 KO). Comparando os tumores de animais WT e Gal-3 KO, não observamos diferenças no padrão de crescimento tumoral, na área necrótica relativa, na proliferação celular e na quantificação de fibras de colágeno. Demonstramos que, embora ambos os grupos desenvolvam tumores, a angiogênese foi inibida em um microambiente desprovido de gal-3. Entretanto, não houve diferença na produção do fator de crescimento endotelial vascular (VEGF). As imagens obtidas in vivo indicaram que gal- 3 também influencia na formação estrutural de vasos adjacentes ao tumor. Além de mediar aspectos morfológicos relacionados à angiogênese, demonstramos que gal-3 também contribuiu para a funcionalidade vascular, pois houve uma redução na velocidade de fluxo sanguíneo nos vasos intratumorais de animais Gal-3 KO. Nossos dados sugeriram que há menos macrófagos no tumor que não expressa gal-3 e, dentre os TAMs, há mais M2 em comparação ao tumor gal-3-positivo. A análise do tecido onde o tumor se desenvolve, na fase inicial da tumorigênese, indicou que a ausência de gal-3 está relacionada a uma maior densidade de macrófagos M2. Considerando que a presença maior de macrófagos M2 nos sarcomas gal-3-negativos não resultou em maior produção de VEGF, mas sim na inibição da angiogênese, nossos resultados apontam para uma participação significativa de gal-3 na mediação da angiogênese pelos macrófagos / As well as normal tissues, tumors require nutrients and oxygen, which are supplied by the associated vasculature that results from the process of angiogenesis. Pro-angiogenic factors are able to attract monocytes and they differentiate into tumor-associated macrophages (TAMs). TAMs commonly exhibit M2 phenotype, which has characteristics considered pro-tumoral, such as angiogenesis promotion and degradation of extracellular matrix. Studies show that galectin-3 (gal-3), a pleiotropic ?-galactosidebinding protein, participates in angiogenesis control and M2 macrophage infiltration into the tumor mass, but little is known about the mechanisms involved. In this work, we established a model of carcinogen-induced sarcoma in wild-type (WT) and gal-3 knockout (Gal-3 KO) mice. Comparing tumors from WT and Gal-3 KO animals, there were no differences in the pattern of tumor growth, relative necrotic area, cell proliferation and collagenous fibers. We demonstrated that, although both groups develop tumors, angiogenesis was inhibited in a microenvironment devoid of gal-3. However, there was no difference in the production of vascular endothelial growth factor (VEGF). The images obtained in vivo indicated that gal-3 also influenced the structural formation of vessels adjacent to the tumor. In addition to mediating morphological aspects related to angiogenesis, we demonstrated that gal-3 also contributes to vascular functionality, since there was a reduction in blood flow velocity in intratumoral vessels from Gal-3 KO animals. Our data suggested that there are fewer macrophages in tumors without gal-3 and, among TAMs, there are more M2 compared to gal-3-positive tumors. Analysis of the tissue where the tumor develops, in early stages of tumorigenesis, indicated that the lack of gal-3 is related to an increased density of M2 macrophages. Since the greater number of M2 macrophages in gal-3-negative fibrosarcomas did not result in increased VEGF production, but inhibited angiogenesis, our results suggest a significant role of gal-3 in regulation of angiogenesis by macrophages

Vnn1 est une pantéthéinase participant à la gestion du stress tissulaire. L’activité pantéthéinase lyse la pantétheine, intermédiaire métabolique dans la synthèse du coenzyme A en cystéamine et pantothénate. Vnn1 est inductible sur des fibroblastes activés et participe à la réparation vasculaire en réponse à l'hypoxie. L’objectif de cette thèse est de déterminer le rôle de Vnn1 dans deux pathologies liées à des dysfonctions myofibroblastiques : la sclérodermie et le fibrosarcome. Dans le cadre d’une collaboration, nous avons montré que Vnn1 est induite sur des fibroblastes sclérodermiques chez la souris et chez l’homme. Des souris Vnn1-/- développent une forme atténuée de la pathologie, avec une réduction de la fibrose et de l’inflammation. Vnn1 pourrait être un marqueur pronostique des formes graves en pathologie humaine. En parallèle, nous avons montré que Vnn1 est exprimée au cours du développement de fibrosarcomes dans un modèle de souris Ink4a/Arf-/-, et se comporte comme un gène suppresseur de tumeur dans ce modèle. Nous avons produit des lignées fibroblastiques Ink4a/Arf-/- qui ont été transformées avec RasV12 en présence de Vnn1 active ou pas. Après greffe, ces lignées produisent des fibrosarcomes. En présence de Vnn1 active, la croissance tumorale est réduite et leur état de différenciation maintenu. De plus, la présence de Vnn1 semble réduire la transition métabolique induite par effet Warburg, amplifiant la glycolyse aérobie et limitant l’activité mitochondriale. Ces résultats montrent que Vnn1 corrèle avec un état activé du myofibroblaste, ce qui est délétère dans le cas de la sclérodermie mais limite la progression tumorale dans le cas des fibrosarcomes. / Vanin-1 (Vnn1) is a pantetheinase which participates in the control of tissue stress. Pantetheinases hydrolyse pantetheines, metabolic intermediates in the synthesis of coenzyme A into cysteamine and pantothenate. Vnn1 is inducible in activated fibroblasts and participates in vascular repair during responses to hypoxic stress. The aim of this thesis was to determine the role of Vnn1 in two pathologies linked to myofibroblasts dysfunction : scleroderma and fibrosarcoma. A collaborative work, have shown that Vnn1 is upregulated in sclerodermic fibroblasts in mouse and in human. Mice Vnn1-/- developed less severe pathology, with a diminution of fibrosis and inflammation. Vnn1 could be a prognostic marker for the severe form of scleroderma. In parallel, we have shown that Vnn1 is expressed during fibrosarcoma development in Ink4a/Arf-/- mice and acts as a tumor suppressor gene in this model. We have generated fibroblastic Ink4a/Arf-/-cell lines which were transduced with RasV12, in presence of a Vnn1 active or not. After engraftment, these cells induced fibrosarcoma. In the presence of the active form of Vnn1, their differentiation into myofibroblasts was maintained and tumor growth was reduced. Moreover, Vnn1 appeared to diminish the metabolic transition induced by the Warburg effect which amplifies aerobic glycolysis, while limiting mitochondrial activity, beneficial for tumor growth. Together, these results show that Vnn1 expression correlates with an activated state of myofibroblasts, which is deleterious in scleroderma but susceptible to limit tumor growth and specifically the development of undifferentiated aggressive fibrosarcoma.

Assim como tecidos normais, tumores possuem uma demanda de nutrientes e oxigênio, suprida através da vasculatura a eles associada que resulta do processo de angiogênese. Fatores pró-angiogênicos são capazes de atrair monócitos, os quais se diferenciam em macrófagos associados a tumores (TAMs). TAMs comumente apresentam fenótipo M2, cujas características são consideradas pró-tumorais, como a promoção da angiogênese e a degradação de matriz extracelular. Estudos indicam que galectina-3 (gal-3), uma proteína pleiotrópica que se liga a ?-galactosídeos, participa do controle da angiogênese e da infiltração de macrófagos M2 na massa tumoral, mas pouco se sabe sobre os mecanismos envolvidos. No presente estudo, utilizamos um modelo de sarcoma induzido por carcinógeno em camundongos selvagens (WT) e knockout para gal-3 (Gal- 3 KO). Comparando os tumores de animais WT e Gal-3 KO, não observamos diferenças no padrão de crescimento tumoral, na área necrótica relativa, na proliferação celular e na quantificação de fibras de colágeno. Demonstramos que, embora ambos os grupos desenvolvam tumores, a angiogênese foi inibida em um microambiente desprovido de gal-3. Entretanto, não houve diferença na produção do fator de crescimento endotelial vascular (VEGF). As imagens obtidas in vivo indicaram que gal- 3 também influencia na formação estrutural de vasos adjacentes ao tumor. Além de mediar aspectos morfológicos relacionados à angiogênese, demonstramos que gal-3 também contribuiu para a funcionalidade vascular, pois houve uma redução na velocidade de fluxo sanguíneo nos vasos intratumorais de animais Gal-3 KO. Nossos dados sugeriram que há menos macrófagos no tumor que não expressa gal-3 e, dentre os TAMs, há mais M2 em comparação ao tumor gal-3-positivo. A análise do tecido onde o tumor se desenvolve, na fase inicial da tumorigênese, indicou que a ausência de gal-3 está relacionada a uma maior densidade de macrófagos M2. Considerando que a presença maior de macrófagos M2 nos sarcomas gal-3-negativos não resultou em maior produção de VEGF, mas sim na inibição da angiogênese, nossos resultados apontam para uma participação significativa de gal-3 na mediação da angiogênese pelos macrófagos / As well as normal tissues, tumors require nutrients and oxygen, which are supplied by the associated vasculature that results from the process of angiogenesis. Pro-angiogenic factors are able to attract monocytes and they differentiate into tumor-associated macrophages (TAMs). TAMs commonly exhibit M2 phenotype, which has characteristics considered pro-tumoral, such as angiogenesis promotion and degradation of extracellular matrix. Studies show that galectin-3 (gal-3), a pleiotropic ?-galactosidebinding protein, participates in angiogenesis control and M2 macrophage infiltration into the tumor mass, but little is known about the mechanisms involved. In this work, we established a model of carcinogen-induced sarcoma in wild-type (WT) and gal-3 knockout (Gal-3 KO) mice. Comparing tumors from WT and Gal-3 KO animals, there were no differences in the pattern of tumor growth, relative necrotic area, cell proliferation and collagenous fibers. We demonstrated that, although both groups develop tumors, angiogenesis was inhibited in a microenvironment devoid of gal-3. However, there was no difference in the production of vascular endothelial growth factor (VEGF). The images obtained in vivo indicated that gal-3 also influenced the structural formation of vessels adjacent to the tumor. In addition to mediating morphological aspects related to angiogenesis, we demonstrated that gal-3 also contributes to vascular functionality, since there was a reduction in blood flow velocity in intratumoral vessels from Gal-3 KO animals. Our data suggested that there are fewer macrophages in tumors without gal-3 and, among TAMs, there are more M2 compared to gal-3-positive tumors. Analysis of the tissue where the tumor develops, in early stages of tumorigenesis, indicated that the lack of gal-3 is related to an increased density of M2 macrophages. Since the greater number of M2 macrophages in gal-3-negative fibrosarcomas did not result in increased VEGF production, but inhibited angiogenesis, our results suggest a significant role of gal-3 in regulation of angiogenesis by macrophages

Cancer remains an elusive killer due, in part, to the suppression of normal immunologic antitumor responses. Normal host (NH) macrophage (Mϕ) populations have tumoricidal effects such as tumor antigen phagocytosis and presentation, and cytokine production. Tumor-infiltrating Mϕs may evade these activities by dysregulating production of immunostimulatory cytokines (including Interleukin [IL]-1β, IL-18, and tumor necrosis factor-α [TNF-α]), by production of antagonistic factors. The restoration of IL-1β, IL-18, and TNF-α production by Mϕs could re-establish antitumor host immune responses. Previous work in our laboratory suggests that tumor distal (TD) Mϕs produce more IL-1β than NH Mϕs when stimulated with IFN-γ and lipopolysaccharide (LPS). We hypothesize that the presence of immunomodulatory factors like IL-10 and TGF-β dysregulate IL-1β production in tumor proximal (TP) Mϕs. Indeed, IL-1β production was downregulated among in situ TP Mϕs. We have proposed that IL-18, a structural homologue to IL-1β was similarly dysregulated in TD and TP Mϕs. IL-18 was enhanced in both distal and proximal Mϕs. Differences in the functions of these cytokines could account for this dissimilarity. TNF-α, another proinflammatory cytokine, followed the dysregulation pattern of IL-1β in our tumor-burdened hosts (TBH), likely because of the similar functions of these cytokines. Because it is a potential vehicle for immunotherapeutic treatment, paclitaxel's action on the immune response (TAXOL™) was investigated. Paclitaxel is a potent Mϕ activator that upregulates a variety of cytokines in an LPS-like manner. Paclitaxel enhanced TD Mϕ production of IL-1β, IL-18, and TNF-α in an LPS-like manner. Production of IL-1β and TNF-α was reduced in TP Mϕs when treated with paclitaxel; however, IL-18 production was enhanced. This difference could be due to the different functions of IL-1β and IL-18. To determine whether production of these cytokines translates into downstream expression of transcription products, IL-12 and nitric oxide (NO) were assayed. NO was enhanced distally, but paclitaxel treatment failed to enhance NO production. When treated with paclitaxel, IL-12 was produced by NH and TD Mϕs. Collectively, these studies suggest that tumor-induced cytokine imbalances compromise antitumor immunity and paclitaxel may reverse this activity. / Master of Science

A laminina contém peptídeos que podem ser liberados por proteólise. Nosso laboratório estuda os efeitos de peptídeos da laminina em biologia tumoral. Neste trabalho, verificamos se C16 (cadeia g1) estimularia invasão e atividade de invadopódios em células de carcinoma epidermóide (CAL27) e fibrossarcoma (HT1080). C16 promoveu aumento na taxa de invasão e atividade de invadopódios em ambas às linhagens celulares, comparado ao peptídeo controle C16SX. Microscopia em time-lapse demonstrou que C16 induz aumento na atividade de invadopódios em função do tempo. C16 estimula fosforilação de Src e ERK 1/2, e inibição da via ERK reduz invasão e atividade de invadopódios relacionados ao peptídeo. C16 conjugado à rodamina foi encontrando decorando a membrana de células CAL27, sugerindo possível interação com receptores. Diminuição dos níveis de integrina b1 reduzem atividade de invadopódios em amostras tratadas com C16. Nossos dados sugerem que C16 regula invasão e atividade de invadopódios em células CAL27 e HT1080, provavelmente por meio de Src, ERK e integrina b1. / Laminin harbors bioactive peptides released upon tumor-induced proteolysis. Our Laboratory has been studying laminin peptides effects in tumor biology. Here we addressed whether C16 (g1 chain) would regulate invasion and invadopodia activity in cell lines from squamous cell carcinoma (CAL27) and fibrosarcoma (HT1080). C16 increased invasion rate and invadopodia activity compared to control peptide (C16SX). Through time-lapse microscopy, we observed that C16 stimulated invadopodia activity overtime. We searched for signaling pathways related to peptide effects. C16 stimulated Src and ERK 1/2 phosphorylation, and ERK signaling cascade inhibition decreased C16-induced invasion and invadopodia. Next, we addressed how C16 would interact with tumor cells. Rhodamine-conjugated C16 was found decorating CAL27 cell membrane, suggesting an interaction with receptors. Knockdown of b1 integrin reduced invadopodia activity of C16-treated cells. We propose that C16 regulates invasion and invadopodia activity of CAL27 and HT1080 cells through Src, ERK and b1 integrin.

A laminina contém peptídeos que podem ser liberados por proteólise. Nosso laboratório estuda os efeitos de peptídeos da laminina em biologia tumoral. Neste trabalho, verificamos se C16 (cadeia g1) estimularia invasão e atividade de invadopódios em células de carcinoma epidermóide (CAL27) e fibrossarcoma (HT1080). C16 promoveu aumento na taxa de invasão e atividade de invadopódios em ambas às linhagens celulares, comparado ao peptídeo controle C16SX. Microscopia em time-lapse demonstrou que C16 induz aumento na atividade de invadopódios em função do tempo. C16 estimula fosforilação de Src e ERK 1/2, e inibição da via ERK reduz invasão e atividade de invadopódios relacionados ao peptídeo. C16 conjugado à rodamina foi encontrando decorando a membrana de células CAL27, sugerindo possível interação com receptores. Diminuição dos níveis de integrina b1 reduzem atividade de invadopódios em amostras tratadas com C16. Nossos dados sugerem que C16 regula invasão e atividade de invadopódios em células CAL27 e HT1080, provavelmente por meio de Src, ERK e integrina b1. / Laminin harbors bioactive peptides released upon tumor-induced proteolysis. Our Laboratory has been studying laminin peptides effects in tumor biology. Here we addressed whether C16 (g1 chain) would regulate invasion and invadopodia activity in cell lines from squamous cell carcinoma (CAL27) and fibrosarcoma (HT1080). C16 increased invasion rate and invadopodia activity compared to control peptide (C16SX). Through time-lapse microscopy, we observed that C16 stimulated invadopodia activity overtime. We searched for signaling pathways related to peptide effects. C16 stimulated Src and ERK 1/2 phosphorylation, and ERK signaling cascade inhibition decreased C16-induced invasion and invadopodia. Next, we addressed how C16 would interact with tumor cells. Rhodamine-conjugated C16 was found decorating CAL27 cell membrane, suggesting an interaction with receptors. Knockdown of b1 integrin reduced invadopodia activity of C16-treated cells. We propose that C16 regulates invasion and invadopodia activity of CAL27 and HT1080 cells through Src, ERK and b1 integrin.