Pre-clinical studies suggest that granulocyte-colony stimulating factor (GCSF) holds promise for the treatment of ischaemic heart disease (IHD). However, its safety and efficacy in this setting, and in particular in patients with chronic refractory 'no-option' IHD, is unclear. Therefore, a clinical study was initiated in 20 such 'no-option' patients, with the aim of assessing the safety and efficacy of both G-CSF administration, and also, that of an intracoronary infusion of G-CSF mobilised CD133+ cells. The study involved initial baseline cardiac ischaemia assessment (symptom based questionnaire, exercise stress test (EST), nuclear Sestamibi (MIBI) and dobutamine stress echocardiographic (DSE) imaging). Stable 'no-option' IHD patients then received open-label G-CSF commencing at 10μg/kg s/c for five days, with an EST on days four and six (to facilitate myocardial cytokine generation and stem cell trafficking). After three months, cardiac ischaemia assessment and the same regimen of G-CSF and ESTs were repeated, but in addition, leukapheresis and then a randomised double-blinded intracoronary infusion of CD133+ or unselected cells were performed. Final cardiac ischaemia assessment was three months thereafter. Eighteen male and two female subjects (mean age 62.4) were enrolled. Eight events occurred that fulfilled pre-specified 'adverse event' criteria: four ischaemic (troponin positive) episodes, two episodes of transient thrombocytopaenia (one profound), one episode of gout and one unscheduled hospitalisation for exhaustion. Troponin was positive on 17 further occasions (all CK-MB negative), however, at these instances angina severity was identical to baseline. Importantly, no adverse event(s) resulted in any detectable long-term adverse sequelae for any subject. From baseline to final follow-up, the administration of two cycles of G-CSF was associated with statistically significant improvements in a range of subjective outcomes, including anginal symptoms, quality of life and EST performance (all p < 0.005). However, the objective MIBI and DSE scans showed only trends towards improvement (all p > 0.1). Compared to unselected cells, an intracoronary infusion of CD133+ cells did not improve either subjective or objective outcomes. In conclusion, administering G-CSF to patients with refractory 'no-option' IHD warrants careful monitoring, but may be performed with safety. A larger, randomised double-blind placebo-controlled trial of G-CSF in these patients appears warranted.

ALVES, Markênia Kélia Santos. Identificação de alterações moleculares associadas à expressão de CD133, CXCR4, CD44 E OLIG2 e metilação em promotor de CDKN2A em tumores astrocíticos. 2014. 89 f. Tese (Doutorado em biotecnologia)- Universidade Federal do Ceará, Fortaleza-CE, 2014. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-09-09T12:10:33Z No. of bitstreams: 1 2014_tese_mksalves.pdf: 2238362 bytes, checksum: f1bbe27ba89548ddbbd8b206939190d7 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-09-27T17:42:58Z (GMT) No. of bitstreams: 1 2014_tese_mksalves.pdf: 2238362 bytes, checksum: f1bbe27ba89548ddbbd8b206939190d7 (MD5) / Made available in DSpace on 2016-09-27T17:42:58Z (GMT). No. of bitstreams: 1 2014_tese_mksalves.pdf: 2238362 bytes, checksum: f1bbe27ba89548ddbbd8b206939190d7 (MD5) Previous issue date: 2014 / Currently, the concept that tumors are cell populations organized in a hierarchically heterogenous way in which stem-cells are relevantly important as these cells have the capacity of self-renew and of generating cell lineages in different phases of differentiation. So that, the identification of stem-cell components is essential to tumorigenesis understanding. Althought neural cell lineage markers have been identified, the association among these markers and neurological tumors is still scarce, and taking in consideration the astrocytomas, the association assessements are verified mainly regarding the glioblastomas. Among these stem cell markers, CD133, CXCR4 and CD44 are related to the glioma formation, migration and growth; on the other hand, OLIG2 is involved in cell destination. So far there are no studies evaluating all these markers together and their relationship to tumor grades. Additionally, specific epigenetic alterations, specially promoter methylation, have been widelly identified in these tumors, leading to gene inativation, mostly involving CDKN2A (p16INK4A protein), a tumor suppressor. Althought this mechanism is pointed as this gene main inactivator, there are still controvertial questions regarding the astrocytomas. In order to evaluate these questions, the present study aimed to determine CDKN2A pattern of methylation and expression and their association to clinicalpathological parameters, and if the presence of progenitor/stem-cells, taking CD133, CXCR4, CD44 and OLIG2 expression in consideration, could define subpopulations of cells which might be used as prognostic markers. So, in a series of 93 astrocytomas of different malignity grades, the expression of CD133, CXCR4, CD44, OLIG2 and p16INK4A was analysed by the imunohistochemistry technique, and the CDKN2A methylation status was assessed by methylation specific PCR (MS-PCR). The data was then associated to tumor grades, localization and other clinicalpathological parameters. The statistic analyses were made using X 2 test, Fisher's exact test, Spearman's correlation, kmeans groupment and principal component analyses, using p<0.05 as statistically significance. The imunopositivity of OLIG2 was predominant (73.1%), followed by CXCR4 (60.2%), CD44 (55.9%) and CD133 (45.2%). The correlation and groupment analyses defined two different population subtypes, a CXCR4(+)CD133(+)CD44(+) subtype and a OLIG2(+) subtype. CXCR4(+)CD133(+)CD44(+) tumors became more frequent as malignity grew. In grade IV, this subtype was significantly more frequent (p=0.008), being also in diffuse tumors. Additionally, CXCR4(+) and CD133(+) tumors were preferentially located in brain hemisferes and in the ventricles, and mostly in aged >30 patients. On the other side, OLIG2(+) tumors were associated to the cerebellum, which is the pylocitic tumor preferential localization. A strong negative correlation between nuclear and cytoplasmatic imunopositivity and promoter methylation in CDKN2A was observed. Also, a negative significant correlation between methylated CDKN2A and patient's age was found; moreover, feminine patients presented a higher frequency of methylated CDKN2A. In conclusion, the presence of stemcell subpopulations in astrocytomas indicates tumoral progression, in which CXCR4, CD133 and CD44 may be potentially used together as prognostic markers. The association between tumor localization and patient's age also corroborates these findings. Additionally, the CDKN2A inactivation by promoter methylation is a frequent event in astrocytomas and it is associated to patient's age and gender. / Tumores são populações celulares heterogêneas hierarquicamente organizadas, cujas células-tronco possuem importância relevante desde que são células com a capacidade de se renovarem e de gerarem linhagens em fases diferentes. Dada a sua importância, a identificação de componentes de células-tronco é essencial para o entendimento da tumorigênese. Apesar de marcadores de linhagem neural terem sido identificados, a associação destes marcadores com os tumores cerebrais ainda é escassa e nos astrocitomas são relacionados principalmente aos glioblastomas. Entre esses marcadores de células-tronco,CD133, CXCR4 e CD44 são relacionados à formação do glioma, migração e crescimento; por outro lado, OLIG2 é envolvido no destino celular. Não existem estudos, até essa data, que avaliam todos esses marcadores juntos e sua relação com grau tumoral. Adicionalmente, alterações epigenéticas específicas, especialmente a metilação em promotor, tem sido identificadas nestes tumores, levando a inativação de genes, com destaque o CDKN2A (proteína p16INK4A), um supressor tumoral. Apesar de esse mecanismo ser apontado como o principal inativador desse gene, em astrocitomas ainda existem questões controversas. Para avaliar essas questões, este estudo objetivou determinar a expressão e padrão de metilação em promotor de CDKN2A e sua associação com parâmetros clinico-patológicos e se a presença de células-tronco/progenitoras, considerando a expressão de CD133, CXCR4, CD44 e OLIG2 poderia definir subpopulações de células que podem ser usadas como marcadores prognósticos. Para isso, em uma série de 93 astrocitomas de diferentes graus de malignidade, foram estudadas a expressão dos marcadores CD133, CXCR4, CD44, OLIG2 e p16INK4A, detectada pela técnica de imunohistoquímica, e o padrão de metilação em promotor de CDKN2A, por PCR específico para metilação (PCR-MS). Os dados foram então associados com grau tumoral, localização e outros parâmetros clinico-patológicos. As análises estatísticas foram realizadas usando o teste do X2, teste exato de Fisher, correlação de Spearman, agrupamento de k-means e análise de componentes principais, com diferenças consideradas significantes com p<0.05. A imunomarcação de OLIG2 mostrou a frequência maior de positividade (73,1%), seguido por CXCR4 (60,2%), CD44 (55,9%) e CD133 (45,2%). Análises de correlação e agrupamento definiram dois subtipos de população de acordo com os marcadores estudados, um subtipo CXCR4(+)CD133(+)CD44(+) e outro OLIG2(+). Tumores CD133, CXCR4 e CD44 positivos aumentaram de acordo com malignidade. No grau IV, este subtipo de tumores [CD133(+)CXCR4(+)CD44(+)] foi significantemente mais frequente (p=0,008) e também nos tumores difusos. Adicionalmente, tumores com CXCR4(+) e CD133(+) foram preferencialmente localizados nos hemisférios cerebrais e nos ventrículos, e a maioria nos pacientes com idade ≥ 30 anos. Por outro lado, tumores OLIG2(+) foram associados com o cerebelo, que é a localização preferencial do astrocitoma pilocítico. Uma forte correlação negativa entre imunomarcação nuclear e citoplasmática e metilação em promotor de CDKN2A foi encontrada. Além do mais, uma correlação negativa significante entre metilação em promotor de CDKN2A e idade foi observada e pacientes do sexo feminino tiveram uma maior frequência significante de CDKN2A metilado em promotor que o sexo masculino. Em conclusão, a presença de subpopulações de células-tronco em astrocitomas é indicativa de progressão tumoral, cujos marcadores CXCR4, CD133 e CD44 podem ser potencialmente usados em conjunto como marcadores prognósticos. A associação com localização do tumor e idade também corroboram esses achados. Adicionalmente, a inativação de CDKN2A por metilação em promotor é um evento frequente em astrocitomas e é relacionada à idade e sexo dos pacientes.

Au sein des tumeurs, y compris pour le carcinome hépatocellulaire (CHC), des sous-populations de cellules néoplasiques ont révélé une grande capacité à initier de nouvelles tumeurs et à induire des métastases. Les premières études sur ces cellules ont rapidement montré que la présence de ces cellules était déterminante dans le développement tumoral et elles ont donc été renommées « cellules souches cancéreuses » (CSCs). Malheureusement les mécanismes impliqués dans la maintenance de ces CSCs ne sont que partiellement compris. Par ailleurs dans le CHC un lien a été établi entre les signaux du facteur de croissance de transformation (Transforming Growth Factor, TGF-ß) provenant du microenvironnement tumoral et certaines populations de cellules cancéreuses dont la présence est corrélée à un faible pronostic. La façon dont TGF-ß peut ainsi établir et modifier un phénotype cellulaire dans le CHC reste néanmoins obscure. La méthylation de l’ADN étant un acteur majeur dans la mise en place des programmes cellulaires, notre but a été de caractériser le méthylome de CSCs hépatiques et son lien avec la capacité de TGF-ß à induire des CSCs. Nous nous sommes appuyés sur l’expression du marqueur CD133 pour définir la population de CSCs hépatiques. Afin comprendre l’importance des marques de méthylation de l’ADN dans les CSCs hépatiques, nous avons dans un premier temps déterminé quelle était la signature des cellules CD133+ au niveau de la méthylation de l’ADN en utilisant des puces de méthylation à grande échelle. Les sites CpG différentiellement méthylés ont montré un enrichissement pour d’une part des voies de signalisation déjà identifiées dans les CSCs et, d’autre part, pour des voies de signalisation associées au processus inflammatoire dont la voie TGF-ß/SMAD. Par la suite, nous avons montré que TGF-ß pouvait induire de façon permanente les cellules CD133+ contrairement à une autre cytokine influente dans le cancer du foie, l’interleukine 6. Cette augmentation de cellules CD133+ induite par TGF-ß est associée à des changements de méthylation de l’ADN sur l’ensemble du génome et qui sont, de plus, maintenus au cours des divisions cellulaires. La comparaison entre les deux méthylomes (liés aux cellules CD133+ et à l’action de TGF-ß) a exposé une signature commune significative indiquant que TGF-ß pourrait promouvoir le phénotype de CSC via le processus de méthylation de l’ADN. Mais nous avons également déterminé qu’une grande partie des effets sur la méthylation induits par TGF-ß était totalement indépendante de l’induction de cellules CD133+. Enfin, nous avons observé que les sites de méthylation sensibles au signal de TGF-ß étaient regroupés de façon significative au niveau de régions « enhancer » qui régulent la transcription des gènes. Par ailleurs, ces sites incluaient également des gènes précédemment identifiés comme cibles de TGF-ß mais aussi des gènes codant pour des acteurs épigénétiques de premier ordre comme les méthyltransférases de l’ADN. Ces résultats constituent la première description d’une signature de méthylation de l’ADN induite par TGF-ß permettant une reprogrammation stable vers un profil épigénétique de CSC hépatiques. / Distinct subpopulations of neoplastic cells within tumors, including hepatocellular carcinoma (HCC), display a pronounced ability to initiate new tumors and induce metastasis. Investigations on theses cells rapidly described them as essential for tumor growth and based on theses observations they have been named “cancer stem cells” (CSCs). Unfortunately, the mechanisms involved in sustaining their programs are only partially known. In HCC, there is an established link between microenvironmental signals from Transforming Growth Factor beta (TGF-ß) and survival of certain cell subpopulations which is results in a bad prognosis. However, how TGF-ß establishes and modifies cell behavior in HCC is not fully understood. As DNA methylation is involved in establishing cellular programs, our aim was to characterize the methylome of putative liver CSCs, and its link to the ability of TGF-ß to induce liver CSCs. We used CD133 expression as a positive marker for liver CSC. To understand the relevance of DNA methylation programs in liver CSCs, we first defined the methylome signature of CD133+ cells in liver cancer cells using methylation bead arrays. Differentially methylated CpG sites were enriched in known pathways related to CSC survival and to inflammation, including the TGF-ß/SMAD pathway. Next, we showed that TGF-ß persistently induces CD133+ cells in opposition to another cytokine related to HCC, interleukin 6. We observed that this increase is associated with genome-wide changes in the methylome induced by TGF-ß and that are perpetuated through cell division. We observed a significant overlap between the CD133+ methylome and the methylome induced by TGF-β, indicating that TGF-ß may induce CSC phenotype through DNA methylation reprogramming. Additionally, we observed genome-wide effects of TGF-ß that are independent of the induction of CD133. Finally, TGF-ß methyl-sensitive sites were significantly concentrated in enhancer regions of the genome, and include well-known targets of TGF-ß, and epigenetic players, such as de novo DNA methyl-transferases. In conclusion our results are the first indication of the ability of TGF-ß to induce genome-wide changes of DNA methylation, leading to a stable switch to a liver cancer stem cell epigenetic program.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Tumores sÃo populaÃÃes celulares heterogÃneas hierarquicamente organizadas, cujas cÃlulas-tronco possuem importÃncia relevante desde que sÃo cÃlulas com a capacidade de se renovarem e de gerarem linhagens em fases diferentes. Dada a sua importÃncia, a identificaÃÃo de componentes de cÃlulas-tronco à essencial para o entendimento da tumorigÃnese. Apesar de marcadores de linhagem neural terem sido identificados, a associaÃÃo destes marcadores com os tumores cerebrais ainda à escassa e nos astrocitomas sÃo relacionados principalmente aos glioblastomas. Entre esses marcadores de cÃlulas-tronco,CD133, CXCR4 e CD44 sÃo relacionados à formaÃÃo do glioma, migraÃÃo e crescimento; por outro lado, OLIG2 à envolvido no destino celular. NÃo existem estudos, atà essa data, que avaliam todos esses marcadores juntos e sua relaÃÃo com grau tumoral. Adicionalmente, alteraÃÃes epigenÃticas especÃficas, especialmente a metilaÃÃo em promotor, tem sido identificadas nestes tumores, levando a inativaÃÃo de genes, com destaque o CDKN2A (proteÃna p16INK4A), um supressor tumoral. Apesar de esse mecanismo ser apontado como o principal inativador desse gene, em astrocitomas ainda existem questÃes controversas. Para avaliar essas questÃes, este estudo objetivou determinar a expressÃo e padrÃo de metilaÃÃo em promotor de CDKN2A e sua associaÃÃo com parÃmetros clinico-patolÃgicos e se a presenÃa de cÃlulas-tronco/progenitoras, considerando a expressÃo de CD133, CXCR4, CD44 e OLIG2 poderia definir subpopulaÃÃes de cÃlulas que podem ser usadas como marcadores prognÃsticos. Para isso, em uma sÃrie de 93 astrocitomas de diferentes graus de malignidade, foram estudadas a expressÃo dos marcadores CD133, CXCR4, CD44, OLIG2 e p16INK4A, detectada pela tÃcnica de imunohistoquÃmica, e o padrÃo de metilaÃÃo em promotor de CDKN2A, por PCR especÃfico para metilaÃÃo (PCR-MS). Os dados foram entÃo associados com grau tumoral, localizaÃÃo e outros parÃmetros clinico-patolÃgicos. As anÃlises estatÃsticas foram realizadas usando o teste do X2, teste exato de Fisher, correlaÃÃo de Spearman, agrupamento de k-means e anÃlise de componentes principais, com diferenÃas consideradas significantes com p<0.05. A imunomarcaÃÃo de OLIG2 mostrou a frequÃncia maior de positividade (73,1%), seguido por CXCR4 (60,2%), CD44 (55,9%) e CD133 (45,2%). AnÃlises de correlaÃÃo e agrupamento definiram dois subtipos de populaÃÃo de acordo com os marcadores estudados, um subtipo CXCR4(+)CD133(+)CD44(+) e outro OLIG2(+). Tumores CD133, CXCR4 e CD44 positivos aumentaram de acordo com malignidade. No grau IV, este subtipo de tumores [CD133(+)CXCR4(+)CD44(+)] foi significantemente mais frequente (p=0,008) e tambÃm nos tumores difusos. Adicionalmente, tumores com CXCR4(+) e CD133(+) foram preferencialmente localizados nos hemisfÃrios cerebrais e nos ventrÃculos, e a maioria nos pacientes com idade &#8805; 30 anos. Por outro lado, tumores OLIG2(+) foram associados com o cerebelo, que à a localizaÃÃo preferencial do astrocitoma pilocÃtico. Uma forte correlaÃÃo negativa entre imunomarcaÃÃo nuclear e citoplasmÃtica e metilaÃÃo em promotor de CDKN2A foi encontrada. AlÃm do mais, uma correlaÃÃo negativa significante entre metilaÃÃo em promotor de CDKN2A e idade foi observada e pacientes do sexo feminino tiveram uma maior frequÃncia significante de CDKN2A metilado em promotor que o sexo masculino. Em conclusÃo, a presenÃa de subpopulaÃÃes de cÃlulas-tronco em astrocitomas à indicativa de progressÃo tumoral, cujos marcadores CXCR4, CD133 e CD44 podem ser potencialmente usados em conjunto como marcadores prognÃsticos. A associaÃÃo com localizaÃÃo do tumor e idade tambÃm corroboram esses achados. Adicionalmente, a inativaÃÃo de CDKN2A por metilaÃÃo em promotor à um evento frequente em astrocitomas e à relacionada à idade e sexo dos pacientes. / Currently, the concept that tumors are cell populations organized in a hierarchically heterogenous way in which stem-cells are relevantly important as these cells have the capacity of self-renew and of generating cell lineages in different phases of differentiation. So that, the identification of stem-cell components is essential to tumorigenesis understanding. Althought neural cell lineage markers have been identified, the association among these markers and neurological tumors is still scarce, and taking in consideration the astrocytomas, the association assessements are verified mainly regarding the glioblastomas. Among these stem cell markers, CD133, CXCR4 and CD44 are related to the glioma formation, migration and growth; on the other hand, OLIG2 is involved in cell destination. So far there are no studies evaluating all these markers together and their relationship to tumor grades. Additionally, specific epigenetic alterations, specially promoter methylation, have been widelly identified in these tumors, leading to gene inativation, mostly involving CDKN2A (p16INK4A protein), a tumor suppressor. Althought this mechanism is pointed as this gene main inactivator, there are still controvertial questions regarding the astrocytomas. In order to evaluate these questions, the present study aimed to determine CDKN2A pattern of methylation and expression and their association to clinicalpathological parameters, and if the presence of progenitor/stem-cells, taking CD133, CXCR4, CD44 and OLIG2 expression in consideration, could define subpopulations of cells which might be used as prognostic markers. So, in a series of 93 astrocytomas of different malignity grades, the expression of CD133, CXCR4, CD44, OLIG2 and p16INK4A was analysed by the imunohistochemistry technique, and the CDKN2A methylation status was assessed by methylation specific PCR (MS-PCR). The data was then associated to tumor grades, localization and other clinicalpathological parameters. The statistic analyses were made using X 2 test, Fisher's exact test, Spearman's correlation, kmeans groupment and principal component analyses, using p<0.05 as statistically significance. The imunopositivity of OLIG2 was predominant (73.1%), followed by CXCR4 (60.2%), CD44 (55.9%) and CD133 (45.2%). The correlation and groupment analyses defined two different population subtypes, a CXCR4(+)CD133(+)CD44(+) subtype and a OLIG2(+) subtype. CXCR4(+)CD133(+)CD44(+) tumors became more frequent as malignity grew. In grade IV, this subtype was significantly more frequent (p=0.008), being also in diffuse tumors. Additionally, CXCR4(+) and CD133(+) tumors were preferentially located in brain hemisferes and in the ventricles, and mostly in aged >30 patients. On the other side, OLIG2(+) tumors were associated to the cerebellum, which is the pylocitic tumor preferential localization. A strong negative correlation between nuclear and cytoplasmatic imunopositivity and promoter methylation in CDKN2A was observed. Also, a negative significant correlation between methylated CDKN2A and patient's age was found; moreover, feminine patients presented a higher frequency of methylated CDKN2A. In conclusion, the presence of stemcell subpopulations in astrocytomas indicates tumoral progression, in which CXCR4, CD133 and CD44 may be potentially used together as prognostic markers. The association between tumor localization and patient's age also corroborates these findings. Additionally, the CDKN2A inactivation by promoter methylation is a frequent event in astrocytomas and it is associated to patient's age and gender

Inhibitor of differentiation 4 (ID4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer (PCa). Recent studies have shown that Id4 is highly expressed in the normal prostate and decreases in prostate cancer due to epigenetic silencing. Id4 knockdown in androgen sensitive LNCaP cells has been shown to lead to castration resistant prostate cancer (CRPC) in vitro and in vivo. Id4-/- mice leads to underdeveloped prostate with PIN like lesions without the loss of Androgen Receptor (AR) expression. In this study we demonstrate that the loss of ID4 expression in PCa cell line LNCaP and DU145 may promote tumorigenesis by promoting stemness. LNCaP cells with stably silenced ID4 ((-)ID4) using retroviral based shRNA and LNCaP transfected with non-specific shRNA were used to perform colony forming assay and prostatosphere formation using matrigel. Expression of cancer stem cell markers was determined using western blotting and immunocytochemistry (ICC). FACS analysis was used to sort stem cells and determine the ID4 expression. Xenograft study was performed on SCID mice using CD133 positive LNCaP cells. LNCaP(-)ID4 and DU145 cells lacking ID4 showed increased holoclone as well as decreased paraclone formation, which are believed to be derived from stem cells and differentiated cells respectively, as compared to non-silencing control in the colony forming assay. There was also an increase in prostatosphere development in the LNCaP (-) ID4 cells indicating that the loss of ID4 is responsible for promoting the LNCaP cells towards cancer stem cells. The results were further validated via western blotting and ICC using known cancer stem cell markers on the holoclones and paraclones formed by these cells. Xenograft study showed that 10,000 cells from CD133 positive LNCaP cells developed tumor on SCID mice. This study reports for the first time that loss of ID4 increases holoclone and prostatosphere formation indicating that Id4 may contribute to promoting stemness in prostate cancer cells.

Most of tumor consists of a heterogeneous population of tumor cells among a tumor initiating and chemo or radiation resistant subpopulation, called cancer stem cells (CSCs), which have become increasingly important new anticancer targets. CD133 has been recently identified as a prominent marker for CSCs in pancreatic and other tumors; however, the signaling cascade of this cancer stem cell marker has not been fully explored. This study shows increased cell proliferation, colony formation, adhesion, and migration following CD133 overexpression in pancreatic ductal adenocarcinoma (PDAC) cells. Signaling studies have indicated that CD133 overexpression increases the epidermal growth factor receptor (EGFR) activation and phosphorylation of PI3K/Akt and MAPK/ ERK pathways. An in vivo xenograft study confirmed that overexpression of CD133 has higher tumorgentic ability than control mice. Molecular studies have found that CD133 physically associates with EGFR and promotes EGFR protein level and its phosphorlyation, which might be critical for PDAC tumor progression and chemoresistance. The data also showed that CD133 overexpression suppresses the EGF mRNA expression, which may imply that CD133 induces EGFR activation through an EGF ligand-independent process. The findings here point to an important mechanism of action for CD133 in PDAC. The EGFR inhibitor has potent anti-CD133 activity, and the current results have important implications for developing targeting CD133 activity as a novel cancer therapy strategy and the inhibitor approach presented here identifies the inhibition of CD133 activity by the EGFR inhibitor and sheds light on developing a new cancer therapeutic that functions by targeting CD133 activity in human cancer.

The enhanced expression of histone deacetylases (HDACs) in a variety of malignancies drew attention to investigate a new category of anti-cancer drugs that are based on the inhibition of those enzymes. Valproic acid (VPA) is a well-known antiepileptic drug that exhibits antitumor activities through inhibition of HDACs class I and IIa. Cancer stem cells (CSCs) have been recognized to drive the tumor growth and progression hence; attention has been given to target this small subpopulation of CSCs rather than the whole bulk tumor cells. CD133 is considered to be a CSC marker in several tumors and its transcription is strongly influenced by epigenetic changes that will be altered upon administration of histone deacetylase inhibitors (HDACi) in cancer treatment. Therefore, we evaluated the epigenetic and cytotoxic effects of treatment with 1 mM VPA in combination with other chemotherapeutics and its influence on the expression of CD133 in human neuroblastoma (NB) cell lines. Our results revealed that addition of VPA to DNA-damaging chemotherapeutics induced a synergistic anti-tumor effect that was associated with caspase-3 dependent induction of apoptosis in UKF-NB-4 cells. This synergism was related to the increase of the acetylation status of histones H3 and H4 and was only produced either by...

To address the unmet clinical need to eradicate treatment-resistant CD133+ cancer stems within tumors, a CSC-targeted radionuclide therapy (TRT) and companion diagnostic imaging probes were developed utilizing CD133-targeting antibodies and antibody fragments. In Chapter 1, background research providing context for the work in this Thesis is presented. In Chapter 2, a CD133-targeting antibody, RW03IgG, underwent radiolabeling with lutetium-177 to synthesize [177Lu]Lu-DOTA-RW03IgG for CD133-TRT. The CD133-TRT was evaluated for pharmacokinetics and treatment of a CD133 expressing human colorectal tumor bearing mouse model. Biodistribution studies on [177Lu]Lu-DOTA-RW03IgG demonstrated notable uptake in the colorectal tumors and off-target organ uptake consistent with previously reported antibody-based TRTs. Confirmation that tumor uptake was mediated by antibody-antigen binding was verified through co-injection with an excess dose of unlabeled RW03IgG. A dose-escalation therapy trial using [177Lu]Lu-DOTA-RW03IgG for treatment of the colorectal cancer mouse model revealed a dose-dependent reduction in tumor growth rate at well-tolerated doses. The decrease in tumor growth rate observed due to [177Lu]Lu-DOTA-RW03IgG treatment, along with an improvement in overall mouse survival, demonstrate the therapeutic efficacy of CD133-TRT. Additionally, histopathological and immunohistochemical (IHC) analyses indicated low off-target organ toxicity and significant anti-tumor effects. These findings suggested the potential for enhanced overall survival benefits through multiple doses. However, results on multiple-dosed CD133-TRT on the tumor growth rate and overall mouse survival were inconclusive. In Chapter 3, an orthotopic patient-derived glioblastoma (GBM) mouse model was developed that replicates anatomical pharmacokinetic challenges and CSC populations observed in patient tumors. Stereotactic engraftment of the patient GBM cells was optimized to reproducibly deliver tumor cells to the thalamus and growth was monitored using bioluminescence imaging. Ex vivo analysis confirmed various key characteristics of patient GBM, including CD133 expression, hypercellularity, and invasiveness. Biodistribution studies on [177Lu]Lu-DOTA-RW03IgG using the PDX GBM mouse model indicate antibody-antigen driven tumor uptake, determined through co-injection an excess dose of unlabeled RW03IgG. Ex vivo autoradiography supported the biodistribution results and showed elevated uptake of [177Lu]Lu-DOTA-RW03IgG in tumor relative to non-tumor bearing brain tissue. Chapters 4 and 5 centered on the development and evaluation of companion diagnostic CD133-targeted immunoPET probes. Chapter 4 specifically explored probes derived from the full antibody, RW03IgG. The probes were synthesized by conjugating RW03IgG with DFO-NCS to produce DFO-RW03IgG at different chelator-to-antibody ratios. The various DFO-RW03IgG conjugates were then radiolabeled with zirconium-89 to obtain [89Zr]-DFO-RW03IgG. Biodistribution studies and PET imaging revealed promising tumor uptake of [89Zr]-DFO-RW03IgG, and it was observed that higher chelator-to-antibody ratios led to increased accumulation in off-target organs. Chapter 5 investigated a probe derived from an scFv-Fc fragment of RW03, [89Zr]-DFO-RW03scFv-Fc. Biodistribution studies and PET images of colorectal tumor-bearing mice administered [89Zr]-DFO-RW03scFv-Fc showed favorable tumor uptake and low off-target organ accumulation. In Chapter 6, a probe for CD133-Photoacoustic Imaging (PAI) was synthesized through conjugation of RW03IgG with IR-783, an organic dye recognized for its favorable photoacoustic properties. Challenges were encountered in isolating the product, (IR-783)-RW03IgG, at high degrees of labeling (DOL) due to product aggregation. In vitro binding assays indicated that (IR-783)-RW03IgG (DOL = 1) maintained a comparable binding affinity to native RW03IgG. In vivo, colorectal tumors in mice administered (IR-783)-RW03IgG (DOL = 1) did not exhibit significant contrast from the background tissue, and the tumor PA signal did not differ significantly compared to tumors in mice administered an IR-783 labeled isotype IgG. The results suggest that a higher concentration of dye is needed within colorectal tumors for effective tumor visualization than what was provided by IR-783-RW03IgG. Chapter 7 investigated the use of Imaging Mass Cytometry (IMC) to simultaneously visualize [177Lu]Lu-DOTA-RW03IgG and multiple tumor biomarkers in tissue specimens collected from colorectal tumor xenograft mice treated with CD133-TRT. IMC showed undetectable concentrations of hafnium-177 (the decay product of lutetium-177) in tumors treated with CD133-TRT. However, lutetium-176 and lutetium-175, sourced from the carrier-added [177Lu]LuCl3 used in the synthesis of [177Lu]Lu-DOTA-RW03IgG, were present at levels sufficient for IMC visualization. The distribution of lutetium-176, representing [177Lu]Lu-DOTA-RW03IgG, within tumors, was imaged concomitantly with CD133, DNA damage markers, and several additional biomarkers that describe elements of the tumor microenvironment. These collective results endorse IMC as a useful tool to assess the distribution of TRT within tumors and uncover changes to the microenvironment in response to treatment. / Thesis / Doctor of Philosophy (PhD) / Targeted radionuclide therapy (TRT) and molecular imaging strategies were developed to aid in the elimination of the rare and particularly resilient Cancer Stem Cell (CSC) population in tumors. A fully human monoclonal antibody and antibody fragments targeting CD133, a molecular biomarker for CSCs, provided the means to deliver radioactive isotopes for therapy and imaging to CD133+ cells in tumors. The therapeutic efficacy of CD133-TRT for treatment of a colorectal cell line-derived xenograft mouse model was promising, and the treatment showed uptake in orthotopic patient derived glioblastoma tumors engrafted in mice. ImmunoPET probes targeting CD133 were optimized and successfully delineated CD133 expressing tumors from background tissue, warranting further evaluation using patient-representative cancer models. A non-invasive CD133-targeting Photoacoustic Imaging (PAI) probe was synthesized through conjugation of the CD133-targeting antibody to an organic dye, IR-783, although further probe optimization is required to provide tumor contrast. Tumor specimens from mice treated with CD133-TRT were assessed by Imaging Mass Cytometry (IMC), which revealed detectable concentrations of carrier isotopes from the therapy in the tumors, implicating the discovery of a powerful new tool for multiplexed single-cell level resolution imaging for cellular-scale analysis of targeted radionuclide therapy. The CSC-therapy and select molecular imaging probes generated in this Thesis warrant further evaluation using patient-representative mouse models of cancer.

No description available.

Brain tumors represent a leading cause of cancer mortality, of which medulloblastoma (MB) and glioblastoma (GBM) represent the most frequent malignant pediatric and adult brain tumors, respectively. The identification of a rare clonal population of cells, termed cancer stem cells (CSCs) or brain tumor-initiating cells (BTICs), as having the ability to initiate, proliferate, and maintain tumor growth has offered a developmental framework for studying MB and GBM. Evidence in support of cell signaling programs carried forward from brain development into oncogenesis have provided opportunities for BTIC-directed therapies targeting the key BTIC property of self-renewal. Given that neural stem cells (NSCs) must maintain a relative balance between self-renewal and differentiation, brain tumorigenesis may be conceptualized as a disease of unregulated BTIC self-renewal. In this work, I aim to demonstrate the re-emergence of self-renewal genes that regulate NSCs in BTICs, use the Wnt pathway as a model by which these genes may be regulated in a context-specific manner, and identify clinically tractable therapies directed at the overall BTIC self-renewal signaling machinery. Specifically, in Chapter 2, I describe the presence of a shared signaling program between NSCs and MB BTICs consisting of Bmi1 and FoxG1. In Chapter 3, I provide evidence in support of a context-specific tumor suppressive function for activated Wnt/β-catenin signaling in MB. Lastly, in Chapter 4, I demonstrate a CD133-AKT-Wnt signaling axis in which CD133 functions as a putative cell surface receptor for AKT-dependent Wnt activation in GBM. Overall, the body of this thesis offers a mechanistic model by which BTICs may be regulated and targeted to impair tumor growth and improve overall survivorship in childhood MB and adult GBM. / Thesis / Doctor of Philosophy (PhD)