Dissertations / Theses on the topic 'Cancer stem cells markers'

The primary treatment for colorectal cancer is surgery. Radiotherapy and chemotherapy, sometimes combined, are also frequently used to diminish recurrence risk. In response to radiation exposure, several cellular signaling cascades are activated to repair DNA breaks, prevent apoptosis and to keep the cells proliferating. Several proteins in the radiation response and cell survival pathways are potential targets to enhance the effects of radiation. The epidermal growth factor receptor (EGFR), which is frequently upregulated in colorectal cancer and exhibits a radiation protective function, is an attractive target for treatment. EGFR is activated by radiation which in turn activates numerous signaling pathways such as the PI3 kinase/AKT cascade, the RAS/RAF/ERK pathway and STAT leading to tumor cell proliferation. EGFR is also believed to interact with proteins in the DNA repair process, such as DNA-PKcs and MRE11. The cytotoxic effect of an affibody molecule (ZEGFR:1907)2, with high affinity to EGFR,  in combination with radiation produced a small, but significant, reduction in survival in a KRAS mutated cell line. However, not in the BRAF mutated cell line. The next step was therefore to target proteins downstream of EGFR such as AKT. There was an interaction between AKT and the DNA repair proteins DNA-PKcs and MRE11 and both AKT1 and AKT2 were involved in the radiation response. The knockout of both AKT isoforms impaired the DNA double strand break rejoining after radiation and suppression of DNA-PKcs increased the radiations sensitivity and decreased the DNA repair further. The AKT isoforms also affected the expression of cancer stem cell markers CD133 and CD44 which are associated with the formation of metastasis as well as radiation and drug resistance. The CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. AKT was also involved in cell migration, cell-adhesion and metabolism. Overall, these results illustrate the complexity in response to radiation and drugs in cells with different mutations and the need for combining inhibitors against several targets such as EGFR, AKT, DNA-PKcs, CD133 or CD44.

This study aimed to evaluate the new immunohistochemical markers related to neuroendocrine differentiation induction and stem cells with prognostic factors and biochemical recurrence in patients submitted to radical prostatectomy. Therefore, patients operated at the Hospital Walter CantÃdio, Federal University of CearÃ, in the period of 2008-2013, underwent clinical and outpatient follow-up, between the years 2008-2016. Biochemical recurrence was evaluated and was correlated with pathological characteristics and immunohistochemical reactions. Chromogranin (neuroendocrine differentiation), Aurora Kinase A (AURKA), N-MYC, C-MYC and CD44s were performad in paraffined material. From 74 patients underwent surgery was obtained the followup of 69, in a median period of 41 (2-89) months. Neoplastic neuroendocrine differentiation was associated with seminal vesicles infiltration (p = 0.032) and stage (p = 0.030). C-MYC was associated with Gleason score (p = 0.001) and seminal vesicles infiltration (p = 0.014). AURKA was expressed in rare cases. N-MYC protein was negative in all patients. CD44s was associated with lower preoperative PSA levels and lower Gleason scores. Biochemical recurrence was observed in 27.0% of patients. Recurrence was associated with serum preoperative PSA, Gleason score, seminal vesicle invasion and staging at least in one form of analysis. There was no significant association between recurrence and neuroendocrine differentiation, C-MYC and CD44s expression. Therefore, immunohistochemical detection of neuroendocrine differentiation, expression of C-MYC and loss of CD44s were related to more aggressive carcinomas (PSA, Gleason, seminal vesical invasion and/or stage), but no association with biochemical recurrence. Classic prognostic factors were affirmed like biochemical recurrence predictores.<br>Este estudo objetivou avaliar novos marcadores imuno-histoquÃmicos relacionados à induÃÃo da diferenciaÃÃo neuroendÃcrina e cÃlulas-tronco com fatores de prognÃstico e recorrÃncia bioquÃmica, em pacientes submetidos à prostatectomia radical. Para tanto, pacientes operados no Hospital Walter CantÃdio, Universidade Federal do CearÃ, no perÃodo de 2008 a 2013, foram submetidos a acompanhamento clÃnico-ambulatorial, entre os anos de 2008 a 2016. Avaliou-se a proporÃÃo daqueles que apresentaram recorrÃncia bioquÃmica, bem como as caracterÃsticas clÃnico-patolÃgicas e a marcaÃÃo em reaÃÃes de imuno-histoquÃmica para cromogranina (diferenciaÃÃo neuroendÃcrina), Aurora quinase A (AURKA), N-MYC, C-MYC e CD44s, em material parafinado. De 74 pacientes submetidos à cirurgia, obteve-se acompanhamento de 69, com tempo de seguimento de 41 (2-89) meses; diferenciaÃÃo neuroendÃcrina na neoplasia se associou com infiltraÃÃo de vesÃculas seminais (p=0,032) e estadiamento (p=0,030). C-MYC associou-se com escore de Gleason (p=0,001) e infiltraÃÃo de vesÃculas seminais (p=0,014). AURKA expressou-se em raros casos. N-MYC foi negativo em todos os pacientes. CD44s se associou com menores nÃveis de PSA prÃ-operatÃrio e menores escores de Gleason. Observou-se recorrÃncia bioquÃmica em 27,0% dos pacientes. RecorrÃncia se associou, em pelo menos uma das formas de anÃlise, com nÃveis sÃricos de PSA prÃ-operatÃrio, escore de Gleason, invasÃo de vesÃculas seminais e estadiamento. NÃo houve associaÃÃo significativa entre recorrÃncia e diferenciaÃÃo neuroendÃcrina, C-MYC e CD44s. Dessa forma, nesse estudo, a detecÃÃo imuno-histoquÃmica da diferenciaÃÃo neuroendÃcrina; a expressÃo de C-MYC e a perda da expressÃo de CD44s relacionaram-se com carcinomas mais agressivos (PSA, Gleason, infiltraÃÃo de vesÃcula seminal e/ou estadiamento), porÃm sem associaÃÃo com a recorrÃncia bioquÃmica; bem como confirma a importÃncia de fatores prognÃsticos considerados como clÃssicos em sÃrie regional de pacientes com cÃncer de prÃstata.

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.

Includes bibliographical references (leaves 142-168) Aim was the purification and identification of the early primitive ectoderm-like (EPL) cell induction signals within the medium conditioned by the human hepatocellular carcinoma cell line HepG2 and the localisation of the signals that induce EPL cell and primitive ectoderm formation.

Platinum-based therapy is the most often used chemotherapeutic agent to treat advanced cases of head and neck cancers. However, only a small fraction of the patient population responds to cisplatin, with a median survival time of less than a year. Currently, there is a lack of clinically employable molecular characterization of the disease beyond HPV status to classify patients who would respond favorably to platinum-based therapy. In this regard, CD24 expression level appears to be a significant molecular phenotype of cisplatin resistance in laryngeal carcinoma. This study demonstrates that CD24 expression level in HNSCC has a linear relationship with cisplatin resistance, and it affects the transcription of critical apoptotic, stem, and drug resistance genes. The knockdown of the CD24 transcript reduces tumor growth rate and increases the overall cisplatin sensitivity in mice xenograft experiments. A retrospective analysis of a cohort of 25 HNSCC patient tumor samples suggests that CD24-high tumors go on to show an unfavorable response to cisplatin treatment. Overall, based on the strength of further clinical analysis, CD24 presents a strong rationale to be utilized as a predictive indicator to stratify head and neck cancer patients for platinum-based therapy. This study also provides a rationale for using CD24 as a therapeutic adjuvant target along with standard cisplatin therapy in head and neck cancers.

Malignant melanoma is one of the most aggressive and treatment resistant cancers. Treatment options include surgery, chemotherapy, immunotherapy and targeted therapies. Unfortunately, the large heterogeneity of melanomas and the presence of stem cells within the tumor mass are the main causes of treatment resistance. Natural compounds have attracted great interest in cancer research for their capability to induce paraptosis, a non-canonical cell death characterized by endoplasmic reticulum (ER) dilatation, mitochondrial swelling, reactive oxygen species (ROS) production and Ca++ overload. Accumulating evidence supported the anticancer effect of tocotrienols (TTs); aim of this study was to investigate whether δ-TT might induce paraptosis in A375 and BLM human melanoma cells. We demonstrated that δ-TT induces paraptosis in melanoma cells, causing ER dilatation, mitochondria swelling and the activation of MAPKs. This effect was accompanied by an impairment of mitochondrial function, resulting in a reduction in OXPHOS levels, oxygen consumption, mitochondrial activity and energy depletion. This dysfunction leads to mitochondrial ROS production and increase in Ca++ levels. We then focused our attention on A375 stem cells. Cancer stem cells (CSCs) are a small tumor subpopulation responsible for chemotherapy resistance and they have been reported to be coupled with metabolic alterations. Melanoma SCs deriving from the A375 cell line are characterized by the expression of the ABCG2 transporter and δ-TT treatment counteracts A375 SCs growth. In this work we observed that melanoma SCs are characterized by higher mitochondrial mass, increased OXPHOS, increased PGC1α expression levels and increased mitochondrial fusion markers. We then analyzed whether δ-TT might affect stem cell mitochondria and we observed that δ-TT significantly impairs mitochondrial protein levels. Finally, preliminary data demonstrate that δ-TT affects mitochondria also in vemurafenib-resistant A375 cells. Taken together, all these data demonstrate that 1) δ-TT induces paraptotic cell death in melanoma cells, involving mitochondrial dysfunction, ROS production and Ca++ overload; 2) mitochondrial mass could be considered a novel marker to identify melanoma SCs; 3) δ-TT targets melanoma SCs affecting mitochondrial functions 4) δ-TT impairs mitochondria in vemurafenib-resistant A375 cells.

Mestrado em Biologia Molecular e Celular<br>Os marcadores de células estaminais constituem excelentes ferramentas de prognóstico para determinar a progressão e sobrevivência de pacientes diagnosticados com cancro da próstata metastático. Dentro deste grupo de marcadores encontra-se o SOX2, um fator de transcrição expresso em lesões metastáticas de carcinomas da próstata resistentes aos androgénios. Em neoplasmas primários da próstata, a expressão de SOX2 encontra-se frequentemente elevada, apresentando uma utilidade semelhante ao fator Gleason, e ao antigénio específico da próstata para o prognóstico numa pequena serie de 30 pacientes. Modelos experimentais em ratinhos apontam que células basais SOX2+ contribuem para a população de células luminais e para a tumorigénese em ratinhos mutantes para a p63 e o PTEN. O SOX2 é um gene cuja expressão é reprimida pelo receptor de androgénios, possuindo um papel essencial na aquisição de fenótipos de resistência à castração. Além disso, encontra-se igualmente envolvido nos processos de aquisição de resistência ao paclitaxel na linha celular PC3 através da via de sinalização PI3K/Akt. No entanto, apesar do seu papel no aparecimento de metástases ser cada vez mais evidente, a sua função e a forma como é modulado neste tipo de lesão permanece ainda por identificar. Após confirmação de um diagnóstico positivo para cancro da próstata, ao paciente podem ser apresentadas várias vias de tratamento, incluindo opções de monotorização ativa, prostectomia radical ou radioterapia. Atualmente, a prostectomia radical constitui uma cirurgia associada a elevadas taxas de mortalidade e a elevados gastos económicos para o sistema de saúde. Além disso, as terapias habitualmente direcionadas para o cancro da próstata interferem significativamente com a qualidade de vida do paciente. Assume-se assim, que o desenvolvimento de novas ferramentas terapêuticas, assim como de diagnóstico, será de extrema urgência. Este projeto tem como objetivo avaliar a possível aplicação do SOX2 no desenvolvimento de novos critérios de diagnóstico, assim como determinar o seu possível envolvimento na progressão de carcinomas da próstata. Especificamente, o principal objetivo deste estudo é compreender qual o papel exato do SOX2 nos processos invasivos, utilizando uma série de casos e linhas celulares de cancro da próstata. Assim, foi possível demonstrar que o silenciamento do SOX2 mediado por siRNAs fomentou uma redução significativa das capacidades migratórias, e ainda, um aumento da morte celular programada nas células da linha PC3, onde a expressão de SOX2 foi silenciada. Verificamos que a expressão de SOX2 em células basais de amostras de cancro da próstata, encontrava-se aumentada em comparação ao tecido adjacente normal, corroborando a hipótese de que o SOX2 poderá estar envolvido na tumorigénese da próstata. Procedeu-se ainda a uma avaliação da relação existente entre a expressão de SOX2 e uma série de critérios clínicos e histopatológicos. Verificou-se que o SOX2 encontra-se significativamente associado a um maior risco de recorrência após tratamento primário, sugerindo que este fator de transcrição poderá desempenhar uma função importante nos mecanismos subjacentes à recorrência tumoral. De um modo geral, a caraterização das atividades do SOX2 ao longo do desenvolvimento, progressão e recorrência de carcinomas da próstata, tornar-se-á um importante passo no sentido de compreender os processos biológicos implícitos nesta condição. Em última análise, tal poderá conduzir à descoberta de novas estratégias promissoras para o tratamento de carcinomas da próstata, baseadas na repressão de SOX2.<br>Stem cell markers are powerful prognostic tools to predict cancer progression and cancer-specific survival in patients with metastatic prostate cancer (PC). Among these is the SRY (sex determining region y)-box 2 (SOX2) gene. SOX2 is expressed in metastatic lesions of androgen resistant human PC. In primary PC, SOX2 was overexpressed and found to be as useful as Gleason and prostate specific antigen (PSA) for prognosis in a small series of 30 patients. Experimental models in mice suggest that SOX2-expressing basal cells in p63 and Pten-null mice contribute to the luminal population and tumorigenesis. SOX2 is an androgen receptor (AR) repressed gene found to promote castration resistant PC (CRPC) phenotypes. Moreover, it is involved in paclitaxel resistance of the PC cell line PC-3 via the PI3K/Akt pathway. Despite its apparently crucial role in metastasis, the actual role of SOX2 and whether or not it might be modulated in bone metastatic lesions of PC has not been well documented. After obtaining a biopsy positive for PC, patients face the option of waiting or performing a radical prostatectomy (RP) or radiotherapy. RP is a high morbidity associated surgery involving also large economical burden to the healthcare system. Indeed, the available therapies regarding the management of PC significantly interfere with the patient wellbeing and thus the development of novel diagnostic and therapeutic tools is mandatory. As such, this project aims at developing SOX2-based diagnostic criteria and to assess its possible role in PC progression. More precisely, the main purpose of this study, using a series of PC cases and cell lines is to study SOX2 and accurately assess its role in invasion. Here, we successfully validated SOX2 as a pivotal player in the early progression and invasion of prostate carcinomas. We have demonstrated that SOX2 silencing mediated by siRNAs stemmed a significant decrease in the migration capacities and an increase in the apoptotic process of SOX2 knocked-down PC3 cells. These results further support the hypothesis that SOX2 might take part of an important transcription program certainty responsible for modulating the establishment of metastasis. We also found that SOX2 was overexpressed in basal-like cells of human PC tissue, and not in normal adjacent tissue, further supporting the possibly involvement of SOX2 in the tumorigenesis of the prostate. In addition, we also evaluated the relationship of the expression of SOX2 with a series of clinical and histopathological criteria and found that it was significantly associated with an increased risk of relapsing after primary therapy, pointing to its possible role in tumour recurrence. In conclusion, we believe that the characterization of the SOX2 activities throughout the development, progression, and relapse of prostate carcinomas will become a major step forward towards understanding the biology underpinning this disorder. Ultimately, such understandings can hopefully lead to the discovery of a SOX2 repressor which could potentially be used for anti-cancer therapy.

Leucine rich repeat containing G-protein coupled receptor 5 (LGR5) is a well-established stem cell marker in the normal intestine. Recent evidence suggests LGR5 is also a marker of cancer stem cells in colorectal tumours. Cancer stem cells propagate and maintain tumours and are hypothesized to be refractory to therapy. Solid tumours frequently experience hypoxia and an unresolved question remains as to how the cancer stem cell population survives this environmental stress. Several regulatory links are known to exist between hypoxic and stem cell signalling pathways. However, the role of hypoxia in regulating LGR5 is yet to be elucidated. Here it is reported that hypoxia down-regulates LGR5 expression. Both protein and mRNA expression is reduced in hypoxia. LGR5 down regulation was observed in cells derived from adenoma, primary carcinoma and metastases, suggesting this process occurs throughout tumourigenesis. LGR5 was found to be re-expressed following re-oxygenation, demonstrating tumour cells ability to switch between expressing LGR5 in normoxic conditions and reducing expression in hypoxia. LGR5 is an established target gene of the WNT signalling pathway and hypoxia has been reported previously to regulate the WNT pathway. Results presented here suggest hypoxic down-regulation of the WNT pathway mediates the hypoxic regulation of LGR5. Topflash WNT reporter activity and expression of a selection of additional WNT target genes decreased in hypoxia in the cell lines tested here. Preliminary ChiP experiments suggest WNT signalling effectors Beta-catenin and TCF4 are lost from the LGR5 promoter in hypoxia. It has been reported previously that HIF-1 interacts with the WNT pathway in hypoxia to down-regulate WNT target gene expression. However HIF-1 does not regulate LGR5 in the cell lines tested here. Highly related HIF-2, though, is critical in the hypoxic regulation of LGR5 in the LoVo cell line and not in other cell lines tested here. The reversible down regulation of stem cell markers during hypoxia may have important implications for targeting cancer stem cells in vivo where tumours are heterogeneous with fluctuating areas of hypoxia.

The high mortality rate associated with epithelial ovarian cancer (EOC) is due to its insidious onset, leading to late diagnosis as well as eventual development of chemoresistance in the majority of patients. Cancer stem-like cells (CSCs) are thought to contribute to development of multi-drug resistant (MDR) tumours partly through their high level of ABC-transporter expression, which enables them to survive chemotherapy. ABC-transporter (MRP1, MRP2, BCRP, Pgp) and putative CSC-marker (ALDH1A1, CD44) expression was therefore evaluated by immunohistochemistry in a paraffin-embedded cohort of 57 high-grade EOC tumours. Typically 9-12% of cells in tumours expressed CD44 / ALDH1A1. These may represent a population enriched for CSCs. ABC-transporters were expressed in 10- 43% of cells on average. Using Spearman rank test, there was no correlation between CSC- marker and ABC-transporter expression, however correlation was observed between many of the ABC-transporters, suggesting existence of highly drug-resistant populations within tumours. Expression of CA125 (a glycoprotein expressed by EOC cells and used clinically to detect EOC relapse) and EpCAM (a cell adhesion molecule expressed by EOC cells) was also investigated. Interestingly, patient tumours with the highest level of EpCAM had longer overall survival by Kaplan-Meier analysis. In addition, increased expression of MRP1 and CD44 predicted poorer patient outcome by Cox regression analysis. In vitro functional assays were also used to identify CSCs. Cells derived from ascites samples had a mean colony-forming efficiency (CFE) or 6.3%, and in unsorted tumours this was 11.4%. Cancer cells were then isolated from primary ascites and tumour samples (using CA125 and EpCAM). On average, self-renewing assays revealed a 2.2-2.4-fold increase in CFE for CA125 or EpCAM positive sorted cells compared to CA125 or EpCAM negative cells. Future studies could characterise these self-renewing populations, which may lead to identification of novel CSC targets for use in EOC treatment.

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina<br>Neural stem/progenitor cells (NSPC) can differentiate into neurons and glial cells in the central nervous system. Interestingly, NSPC biology is being applied to the study of human brain tumours, since these cells share some common features with glioma cells. However, it is not known the developmental stage with more similarities to glioma cells, or the most susceptible to malignant transformation. We aimed to identify the stage(s) in the NSPC differentiation process towards astrocytes where cells acquire phenotype characteristics comparable to glioma cells. NSPC that were obtained from E15 mouse brain, were grew as neurospheres (NS) and induced to astroglial differentiation until 7 days in vitro (DIV). After the cellular characterization of NS and differentiating cells, tumour-related factors were evaluated and their behavior compared to the one of GL261 mouse glioma cells. Astroglial differentiation led to a decrease in progenitor cells, as expected. Multidrug resistance-associated protein 1 expression decreased and autophagy marker increased with differentiation. The vascular endothelial growth factor (VEGF), matrix metalloproteinases and S100B protein increased until 2/3 DIV, while the 1 DIV cells showed the highest migratory potential towards the chemotactic VEGF or GL261-conditioned media. Comparison of data with glioma cells characteristics point to the first and second days of NSPC differentiation to astrocytes as the stages closing matching GL261 cells, and likely the most vulnerable to malignancy transformation.

Abstract MSCs are the most studied adult stem cells, as they can be isolated from almost any tissue, they show a good self-renewal capacity in vitro and they also possess good “plasticity”(jiang nature). Oddly, MSCs are identified and defined by a combination of markers that are not distinctive, as they are shared by other cells including fibroblasts. Therefore, pure populations of MSCs cannot be isolated, as they are always contaminated by other adult cells that often do not possess stem cell plasticity. Thus, it would be very desirable to discover novel cell surface markers that would allow to discriminate MSCs from other cells. Moreover, new ways to control and promote cell differentiation are needed. Thus, it becomes crucial to search for new key molecules that can be modulated to increase MSCs differentiation toward the desired tissue or inhibit the differentiation when cells have to remain in an undifferentiated state. In this direction, we focused our attention on SLs, a family of lipids found in the outer leaflet of the plasma membrane and involved in many cell signaling pathways. Therefore, main aims of this work were: 1. to investigate the possible use of SLs as new surface markers for the identification, characterization and possibly isolation of human bone marrow MSCs; 2. to investigate the involvement of SLs in the preservation of the undifferentiated state of MSCs during in vitro culturing 3. to assess the possible role of SLs in the differentiation processes of MSCs upon opportune stimuli.

Epithelial Ovarian Cancer (EOC) is a very malignant neoplasm, accounting for 5% of cancer mortality in women. Although progress has been made in EOC treatments by improved debulking surgery and platinum-taxane regimens, the 5-year survival rate of advanced-stage EOC remains below 30%. This poor prognosis relies on the one hand on the late diagnosis and on the other on the chemo-resistance occurring after only a few months from the completion of treatment. The reasons for recurrence and cancer drug resistance remain uncertain. Recent evidence suggests that EOC, akin most tumors, contains a tiny population of cells, named cancer stem cells (CSC), probably responsible for chemotherapy resistance and tumor recurrence. Ovarian CSC are characterized by the co-expression of two surface markers: CD44 (hyaluronic acid receptor) and CD117 (stem cell factor receptor or c-kit). Recently, our research group demonstrated that ovarian CD44/CD117 co-expressing cells, which represent 1-2% of cancer cells from ascitic effusions of EOC-bearing patients, are endowed with canonical stemness properties and are able to resist in vitro and in vivo glucose starvation. We reported that this glucose deprivation resistance is mostly due to the ability of ovarian CSC to privilege oxidative phosphorylation, rather than the aerobic glycolysis (Warburg effect) exploited by the non-stem tumor bulk. However, independently of CSC fraction, our experiments also highlighted that not all the analyzed EOC samples presented a similar glucose addiction. Thus, investigating this issue and its related metabolic aspects is the first aim of the current project. Concerning this aim, we showed that tumor cells from EOC patients can be categorized, according to their in vitro viability under glucose starvation, into glucose deprivation-sensitive (glucose-addicted, GA) and glucose deprivation-resistant (glucose non-addicted, GNA). Although deregulated glucose metabolism is usually observed in cancer, whether this metabolic trait influences response to or is modulated by cytotoxic drugs is unknown; therefore, we addressed the possible correlation of these glucose addiction profiles with the patient response to platinum (PLT) regimens. In this regard, when EOC cells were cultured in the absence of glucose, all samples from PLT-sensitive patients felt into the GA group; compared to GNA samples, they disclosed higher expression of glucose metabolic enzymes, higher proliferation rates and in vitro sensitivity to PLT, as well as reduced multi-drug resistance pump expression. On the other hand, the samples derived from PLT-resistant patients felt into the GNA category. The close association between PLT sensitivity and glucose metabolic profile was confirmed in a xenograft model, where a stringent parallelism between PLT sensitivity/resistance and glucose metabolism was identified. Finally, in a cohort of naïve EOC patients categorized as GA or GNA at diagnosis, Kaplan Meier curves showed that the GA phenotype was associated with significantly better progression-free survival, compared to GNA patients. Overall, these results suggest that in vitro glucose addiction of EOC cells could be regarded as a reliable marker to predict the patient response to platinum regimens. Investigating the molecular traits leading to the distinctive glucose metabolism of EOC samples is the second aim of this project. In this regard, microRNA (miRNA), that are small non-coding RNA molecules, represent a promising field, in view of their ability to modulate many genes and pathways. Moreover, their involvement in cancer development and progression has already been reported in ovarian cancer, and recently miRNA also emerged to regulate cell metabolism in several normal and cancer tissues. Thus, we performed a miRNA profile on EOC patient-derived samples, comparing both GA versus GNA samples and CSC versus non-CSC, in order to establish whether the miRNA signature behind the metabolic differences of EOC samples is associated with the total tumor bulk or rather with a specific cell fraction. The data did not reveal any miRNA differentially expressed in GA compared to GNA cells, but several miRNA resulted significantly deregulated in CSC versus non-CSC. We focused our attention on mir-602, which was found up-regulated in CSC; indeed, despite little information about this miRNA, its target Casein Kinase 1 Delta (CSNK1D), which displays a key role in cell proliferation and asymmetric division, seemed very interesting in view of its involvement in breast cancer progression. We demonstrated that CSNK1D is down-regulated in CSC, according to mir-602 over-expression. Moreover, the in vitro inhibition of mir-602 reduced the expression of the major stemness-associated genes in CSC, suggesting that mir-602 could regulate some cell stemness pathways. Since none of the analyzed stemness genes is directly targeted by mir-602, it is reasonable to advance that mir-602 could indirectly activate the expression of such genes through its inhibitory function on CSNK1D; thus, we propose that cell stemness signaling is inhibited by casein kinase, whose translation is in turn repressed by mir-602. Although a few further experiments are needed to validate our idea, this project highlights a possible miRNA-mediated regulatory mechanism of cell stemness features in EOC.<br>Il tumore ovarico di tipo epiteliale (EOC – Epithelial Ovarian Cancer) rappresenta la prima causa di morte per neoplasia ginecologica. Sebbene la chirurgia e la chemioterapia a base di carbo/cis-platino abbiano migliorato la prognosi delle pazienti affette da tale carcinoma, il tasso di sopravvivenza a 5 anni dalla diagnosi di EOC in stadio avanzato rimane inferiore al 30%. Tale elevata mortalità è riconducibile alla diagnosi in fase tardiva ed all’insorgenza di resistenza alla chemioterapia di prima linea, che porta a frequenti ricadute entro pochi mesi dalla conclusione dei trattamenti. Le cause della comparsa di fenomeni di resistenza alla terapia, e della conseguente ricorrenza del tumore, è ancora incerta. Recenti studi hanno indicato che la crescita del carcinoma epiteliale dell’ovaio è sostenuta da una minima popolazione di cellule, dette cellule tumorali staminali (CSC - Cancer Stem Cells), probabilmente responsabili della ricomparsa del tumore al termine delle sedute farmacologiche. Un consenso generale supporta l’idea che l’eliminazione di questa popolazione rappresenti uno dei più importanti obiettivi della terapia anti-tumorale. Tuttavia, si ha ancora una scarsa conoscenza dei meccanismi che conferiscono un vantaggio di sopravvivenza alle CSC sulle cellule tumorali non staminali, rendendo così arduo lo sviluppo di terapie mirate anti-CSC. Le CSC del carcinoma epiteliale ovarico sono caratterizzate dall’espressione di due marcatori di superficie cellulare: CD44 (recettore dell’acido ialuronico) e CD117 (c-kit o recettore del fattore cellulare staminale SCF). Recentemente, il nostro gruppo di ricerca ha dimostrato che le cellule ovariche co-esprimenti CD44 e CD117, che rappresentano l’1-2% delle cellule tumorali provenienti dall’ascite delle pazienti affette da EOC, possiedono le canoniche proprietà staminali e sono in grado di sopravvivere in vitro ed in vivo alla deprivazione di glucosio. Abbiamo inoltre osservato che tale resistenza all’assenza di glucosio è principalmente dovuta alla capacità delle CSC, contrariamente alle cellule tumorali non staminali, di privilegiare la fosforilazione ossidativa anziché la glicolisi aerobica (Warburg Effect). Tuttavia, indipendentemente dalla frazione delle cellule tumorali staminali, l’analisi comparativa tra i diversi campioni di EOC ha evidenziato che non tutti presentano la stessa dipendenza dai glucidi; per alcuni, infatti, pochi giorni in vitro senza glucosio sono sufficienti a ridurre significativamente la vitalità cellulare, mentre per altri lo stesso effetto è ottenibile solo dopo molte settimane di coltura nelle stesse condizioni. Dunque, approfondire tale questione e gli aspetti metabolici ad essa correlati è il primo scopo di questo progetto. A tal proposito, sulla base della vitalità cellulare in condizioni di coltura senza glucosio, abbiamo potuto suddividere le cellule derivanti da asciti di pazienti con EOC in due categorie: sensibili alla deprivazione di glucosio (GA – Glucose-Addicted) e resistenti a tale deprivazione (GNA – Glucose Non-Addicted). Sebbene variazioni nella regolazione del metabolismo del glucosio siano state frequentemente osservate nei casi di neoplasia, non è ancora noto se questo diverso tratto metabolico influenzi la risposta dei pazienti alle terapie, o se sia da queste modulato. Pertanto, abbiamo deciso di ricercare una eventuale correlazione tra i diversi profili di dipendenza dal glucosio da noi riscontrati e la risposta dei pazienti al trattamento a base di carbo/cis-platino. Infatti, da un punto di vista clinico, i pazienti vengono categorizzati come platino-resistenti o platino-sensibili, a seconda che la neoplasia recidivi entro od oltre i 6 mesi, rispettivamente, dalla fine della chemioterapia di prima linea. I nostri esperimenti hanno rivelato che, quando le cellule di EOC vengono coltivate in assenza di glucosio, tutti i campioni provenienti da pazienti platino-sensibili ricadono all’interno del gruppo GA; confrontati con i campioni GNA, i GA mostrano una maggiore produzione degli enzimi del metabolismo glucidico, un maggior tasso di proliferazione, e una minore espressione delle pompe cellulari per l’espulsione dei farmaci. Parallelamente, i campioni derivanti dai pazienti platino-resistenti rientrano nella categoria GNA. La stretta associazione tra la sensibilità ai chemioterapici e il profilo cellulare di utilizzo del glucosio è stata confermata in un modello murino di xenotrapianti, nel quale è stato identificato uno stringente parallelismo tra risposta al platino e metabolismo glucidico. Infine, in una coorte di pazienti non chemio-trattate affette da EOC, le quali erano state categorizzate come GA o GNA alla diagnosi, le curve di Kaplan Meier hanno messo in luce che il fenotipo GA, rispetto a quello GNA, è associato con un maggior periodo di sopravvivenza senza recidive, in modo statisticamente significativo. Nel complesso, questi dati suggeriscono che il grado di dipendenza dai glucidi delle cellule di EOC, osservabile in vitro, può rappresentare un valido marcatore per predire la risposta dei pazienti alla chemioterapia a base di platino. Analizzare il tratto molecolare che determina il peculiare metabolismo glucidico dei campioni di EOC costituisce il secondo obiettivo del nostro progetto di ricerca. A tal riguardo, i microRNA (miRNA), ossia piccole molecole di RNA non codificante, rappresentano un promettente settore di studio, in qualità della proprietà di queste strutture molecolari di regolare molti geni e vie di segnale. Inoltre, il loro coinvolgimento nello sviluppo e nella progressione tumorale è già stato dimostrato per il carcinoma ovarico, e recentemente i miRNA sono risultati essere importanti modulatori del metabolismo cellulare in molti tessuti normali e neoplastici. Pertanto, il nostro gruppo di ricerca ha prodotto un profilo di espressione di miRNA su cellule derivanti da pazienti affette da EOC, confrontando sia campioni GA contro GNA, sia CSC contro non-CSC; il nostro fine è stabilire se il pattern di miRNA alla base delle differenze metaboliche tra i campioni di EOC sia associato alla totale massa tumorale o piuttosto ad una specifica frazione cellulare neoplastica. Questi dati non hanno rivelato alcun miRNA differentemente espresso tra cellule GA e GNA; tuttavia, molti miRNA sono risultati deregolati nelle CSC rispetto alle non-CSC. Noi ci siamo focalizzati sul mir-602, up-regolato delle CSC; infatti, nonostante la scarsa conoscenza su questo miRNA, il suo target chinasi Caseina 1 Delta (CSNK1D), che detiene un ruolo chiave nella proliferazione cellulare e nella divisione asimmetrica, ci è parso molto interessante in virtù del suo già dimostrato coinvolgimento nella progressione del carcinoma mammario. In questo contesto, i nostri esperimenti hanno dimostrato che CSNK1D è down-espressa nelle CSC, in accordo alla up-modulazione del mir-602. Inoltre, l’inibizione in vitro del mir-602 riduce nelle CSC l’espressione della maggior parte dei geni associati alle proprietà staminali, suggerendo che il mir-602 potrebbe controllare alcune delle vie di segnale correlate alla staminalità. Dato che nessuno dei geni di staminalità analizzati si lega direttamente al mir-602, appare ragionevole supporre che suddetto miRNA possa indirettamente attivare l’espressione di tali geni tramite la sua funzione inibitoria su CSNK1D. Dunque, secondo la nostra ipotesi, le caratteristiche staminali sarebbero inibite dalla chinasi Caseina, la quale sarebbe a sua volta repressa dal mir-602. Nonostante molti altri esperimenti siano necessari per confermare questa nostra teoria, il progetto qui presentato mette in rilievo la possibile esistenza di un meccanismo di regolazione, mediato dal mir-602, responsabile delle proprietà di staminalità delle cellule del carcinoma epiteliale ovarico.

Prostate cancer is a leading cause of cancer-related death in men, and while many men diagnosed with the disease will have an indolent clinical course, 20-25% of men will experience disease recurrence which is invariably lethal. There is an urgent need for prognostic biomarkers that will predict disease recurrence and risk-stratify patients upon diagnosis, allowing for personalised therapies. This thesis attempts to identify new prognostic biomarkers for prostate cancer and investigates their patterns of protein expression in human primary prostate tumour tissue. Cancer stem cells are cancer cells thought to be uniquely capable of self-renewal and tumorigenicity, and may have a role in tumour recurrence. Using a literature searching approach, potential biomarkers related to stem cells, cancer stem cells or recurrence in prostate cancer were identified, and ALDH7A1, BMI1, SDC1, MUC1-C, Nestin and ZSCAN4 were chosen for investigation. An in silico approach was also used for biomarker identification, with RS1 and SLC31A1 selected as their mRNA was found to be upregulated in recurrent tumours. The expression patterns of all 7 potential biomarkers were examined by immunohistochemistry on prostate tumour tissue and benign tissue from prostate biopsies and prostatectomies. BMI1, ALDH7A1, MUC1-C and Nestin showed no relationship to recurrence or other clinical features. RS1 protein levels increased in patients with recurrence within 5 years, negatively correlated with AR expression, and a meta-analysis showed that the RS1 gene was amplified in up to 32% of castration-resistant prostate tumours. ZSCAN4 was heterogeneously expressed in a subset of 26% of prostate tumours with unclear characteristics and was not expressed in benign tissue, but was not associated with recurrence. Finally, SDC1 expression was lost in tumour epithelium, but a population of unidentified SDC1-expressing cells were found in the stroma of a third of tumours, and an increased burden of these cells was associated with primary Gleason pattern 5 tumours. These cells do not overlap with common epithelial, mesenchymal or stromal lineages, but may be migratory. In summary, the data presented in this thesis identifies 3 potential new biomarkers for prostate cancer, and provides the basis for future characterisation of their wider roles in the disease.

Thesis (Ph.D.)--Boston University<br>Each individual breast tumor contains a heterogeneous mix of cancer cells. Recent evidence suggests that the cellular heterogeneity inherent to breast cancer, which underlies metastasis, resistance to treatment and disease recurrence, can be driven by a distinct population of tumor cells referred to as cancer stem cells. However, the extracellular cues and intracellular signaling pathways that cancer stem cells rely on remain largely unknown. The aim of this dissertation was to investigate the functional relevance of tumor cell-derived periostin, a secreted extracellular matrix (ECM) molecule, to cancer stem cells and determine the pathways activated downstream of periostin through the integrin αvβ3 receptor. The rationale for this work was based on correlative evidence that connected periostin and integrin αvβ3 expression to numerous phenotypes associated with cancer stem cells including residence in a cell state associated with epithelial-mesenchymal transition (EMT), mammosphere formation and expression of the CD44+/CD24- surface phenotype. Multiple breast cancer cell lines with knockdown of either periostin or integrin β3 showed characteristics suggestive of a reduced cancer stem cell population such as impaired mammosphere formation and an inability to maintain a subpopulation of aldehyde dehydrogenase (ALDH)-positive cancer cells, implying that this autocrine/paracrine signaling axis is required to maintain breast cancer stem cells. Furthermore, this axis was found to be expressed in a subset of basal-like breast cancer cell lines. Additionally, high periostin expression was associated with a poor prognosis in patients with estrogen receptor (ER)-negative breast cancer. Gene expression analysis revealed that breast cancer cells with knockdown of periostin exhibited significant suppression of a distinct cytokine network, which includes IL-6 and IL-8, two cytokines that have been linked to the regulation of cancer stem cells. In a different cellular context, periostin could regulate the transcription of multiple WNT ligands. Thus, periostin serves as a critical regulator of the cancer stem cell state and acts through integrin signaling to control the production of other secreted factors that can also modulate cancer stem cells. These findings suggest that basal-like breast cancers can establish a microenvironmental niche supportive of cancer stem cells, which might be relevant as a biomarker or therapeutic target.

The discovery of tumor-initiating cells endowed with stem-like features, and therefore referred to as cancer stem cells (CSCs), has added a further level of complexity to the pathobiology of neoplastic diseases. This uncommon cellular subpopulation has been connected with tumor initiation, metastatization and treatment failure. CSCs are protected against standard medical treatments by multiple mechanisms including the abnormal activation of both DNA damage repair signals and canonical survival pathways. Moreover, this cellular subset relies on dedicated signals such as self-renewal-linked molecular circuits. Therefore, the exact definition of the target population and the identification of molecular networks differentially activated in CSCs compared with their differentiated progeny are crucial for an optimal pre-clinical development of molecular targeted agents. We isolated breast CSCs from surgically resected primary breast tumors. A first round of experiments was designed in order to determine whether our clones met the operative criteria to be defined as CSCs. In particular, all these clones possessed: i) the expression of a unique repertoire of markers common to stem and progenitor cells, ii) an unlimited growth in vitro using media optimized for stem cell cultures and iii) the ability to reproduce the parental tumor upon injection in immunocompromised mice. Afterword, the metastatic potential of genetically engineered BCSCs was compared with that of differentiated breast cancer cells (dBCCs) . Upon both intracardiac and orthotopic injection the undifferentiated pool was able to generate distant metastases and to recapitulate the dissemination pattern of the human disease, while dBCCs failed to generate distant lesions. Afterwards, high-throughput assays have been exploited in order to define molecular mechanisms underlying this differential metastatic proclivity. In particular, most up- and down-regulated genes were evaluated for their convergence on canonical signal pathways/biological functions. BCSCs displayed higher levels of DNA repair-linked effectors such as BRCA1, ATR, ATM and Chk1, higher level of the pro-tumorigenic and pro-metastatic protein c-MET and, finally, they were characterized by lower levels of physiological Wnt inhibitors (Dkk family members). Therefore, we identified three different pathways/functions whose hyper-activation seems to be correlated with the metastatic ability of BCSCs. Notably, molecular effectors of the above-mentioned pathways can be pharmacologically antagonized by experimental targeted agents.

Small cell lung cancer (SCLC) is a highly aggressive malignancy with extreme mortality and morbidity. Although initially chemo- and radio-sensitive, almost inevitable recurrence and resistance occurs. SCLC patients often present with metastases, making surgery not feasible. Current therapies, rationally designed on underlying pathogenesis, produce in vitro results, however, these have failed to translate into satisfactory clinical outcomes. Recently, research into cancer stem cells (CSCs) has gained momentum and form an attractive target for novel therapies. Based on this concept, CSCs are the cause of neoplastic tissue development that are inherently resistant to chemotherapy, explaining why conventional therapies can shrink the tumour but are unable to eliminate the tumour completely, leading to eventual recurrence. Here I demonstrate that SCLC H345 and H69 cell lines contain a subset of cells expressing CD133, a known CSC marker. CD133+ SCLC sub-population maintained their stem cell-like phenotype over a prolonged period of culture, differentiated in appropriate conditions and expressed the embryonic stem cell marker Oct-4 indicating their stem-like phenotype. Additionally, these cells displayed augmented clonogenic efficacy, were chemoresistant and tumorigenic in vivo, distinct from the CD133- cells. Thus, the SCLC CD133 expressing cells fulfil most criteria of CSClike definition. The molecular mechanisms associated with CD133+ SCLC chemoresistance and growth is unknown. Up-regulated Akt activity, a known promoter of resistance with survival advantage, was observed in CD133+ SCLC cells. Likewise, these cells demonstrated elevated expression of Bcl-2, an anti-apoptotic protein compared to their negative counterpart explaining CD133+ cell chemoresistance phenotype. Additionally, CD133+ cells revealed greater expression of neuropeptide receptors, gastrin releasing peptide (GRP) and V1A receptors compared to the CD133- cells. Addition of exogenous GRP and arginine vasopressin (AVP) to CD133+ SCLC cells promoted their clonogenic growth in semi-solid medium, illustrating for the first time neuropeptide dependent growth of these cells. A novel peptide (peptide-1) was designed based on the known structure of the substance P analogues that have shown benefit in animal models and in early clinical trials. This compound inhibited the growth of SCLC cells in in vitro with improved potency and stability compared to previous analogues and reduced tumorigenicity in vivo. Interestingly, peptide-1 was more effective in CD133+ cells due to increased expression of neuropeptide receptors on these cells. In conclusion, my results show that SCLC cells retain a sub-population of cells that demonstrate CSC-like phenotype. Preferential activation of Akt and Bcl-2 survival pathways and enhanced expression of neuropeptide receptors contribute to CD133+ SCLC chemoresistance and growth. Therefore, it can be proposed that CD133+ cells are the possible cause of SCLC development, treatment resistance and disease recurrence. Despite being chemoresistant, CD133+ cells demonstrated sensitivity to peptide-1. The identification of such new analogue that demonstrates efficacy towards resistant CD133+ SCLC cells is a very exciting step forward in the identification of a potential new therapy for resistant disease.

Cancer stem cells (CSC) are thought to be responsible for the initiation, propagation and tumour re-occurrence. CSC have been identified in most solid and haematological cancers and account for ~1% of the total cell population. Culturing cancer cell lines in monolayers enriches for the most dominant subpopulation which in most cases does not represent the slow dividing CSC population. We investigated the expression of CSC markers in 2D vs. 3D cell culture with the aim of identifying CSC-like cells via a nanog-reporter cell line and enable the subsequent targeting of these cells with CSC-targeting agents. SUM159 and MCF7 cell lines cultured in 3D cell culture significantly enriched for the CD44+/CD24- breast cancer stem cell phenotype when compared against 2D cell culture (p < 0.05 and 0.001 respectively) and also enriched for expression of CD181 (p < 0.05 and 0.001 respectively). However, this method did not enrich for the prostate CSC with the CD44+/CD133+ phenotype in PC3, DU145 and LNCAP cells. Using reporter cell lines to further identify the CSC population in SUM159 cell line that express GFP in response to Nanog (NRE-GFP), found that these cells were GFP+ve in the absence of Nanog protein. As these reporters were selected based on GFP that was supposedly Nanog driven other mechanistic studies were examined to determine how GFP is expressed in the NRE-GFP and control cell line. It was found that GFP could be induced in apoptosis, CSC enriching medium, hypoxia and by inhibiting the proteasome in the absence of Nanog protein. It was concluded that reporter cell lines that respond to a response element may not identify the CSC population as other factors can induce GFP expression. Compounds related to Withaferin A have been proposed to specifically target CSCs. Prostate and breast cancer cell lines cultured in 2D and 3D were treated with a novel withanolide derivative (LG-02) and Withanilode E to determine if these compounds were more effective at inducing cell cycle arrest and apoptosis using flow cytometry and microscopy. It was determined that LG-02 and WE primary mode of action is cell cycle inhibition and both compounds are more potent cell cycle inhibitors than Withaferin A. G1 phase accumulation was observed in the SUM159, PC3 and LNCAP cell lines and G2/M phase accumulation in the DU145 cell line. Cell cycle arrest was inconclusive in the MCF7 cell line. An apoptotic morphology was only significantly induced at higher concentrations in MCF7, PC3, DU145 and LNCAP. Withanolide derivatives also target the androgen response pathway, demonstrated by a decrease in PSA and androgen receptor in prostate cancer cell lines. LG-02 slowed growth of breast cancer cell lines cultured in 3D and inhibited spheroid formation of prostate cancer cell lines, however the androgen-dependent cell line LNCAP was consistently able to form 3D colonies, most likely via pAkt activation. We conclude that 3D cell culture does enrich for the CSC population in breast cancer cell lines but using a reporter cell line expressing GFP under the control of a NRE is not a suitable model for identifying the CSC population for subsequent drug treatment. In addition, Withanolide derivatives have potential anti-tumour activity and may represent a novel class of anti-androgenic agents.

The generation of induced pluripotent stem cells (iPSCs) presents a challenge to normal developmental processes. The low efficiency and heterogeneity of most methods have hindered understanding of the precise molecular mechanisms promoting, and roadblocks preventing, efficient reprogramming. While several intermediate populations have been described, it has proved difficult to characterize the rare, asynchronous transition from these intermediate stages to iPSCs. The rapid expansion of a minor population of reprogrammed cells can also obscure investigation of relevant processes. Understanding of the biological mechanisms essential for successful iPSC generation requires both accurate capture of cells undergoing the reprogramming process and identification of the associated global gene expression changes. Here we demonstrate that reprogramming follows an orderly sequence of stage transitions marked by changes in cell surface markers CD44 and ICAM1, and a Nanog-GFP reporter. RNA-sequencing (RNA-seq) analysis of these populations demonstrates two waves of pluripotency gene up-regulation, and unexpectedly, transient up-regulation of multiple epidermis-related genes, demonstrating that reprogramming is not simply the reversal of normal developmental processes. This novel high-resolution analysis enables the construction of a detailed reprogramming route map, and this improved understanding of the reprogramming process will lead to novel reprogramming strategies.

The use of stem cells is a promising therapeutic approach for tissue engineering by their ability to boost tissue regeneration, and to model in vitro human genetics disorders since it provides continuous supplies of cells with differentiation potential. Our study has been focused in the identification of molecules or mechanisms that could contribute to a better osteogenesis in mesenchymal stem cells (MSC). To achieve our goals we have explored the osteopontential differences of stem cells from different sources. In this regard, we have observed that MSCs from human exfoliated deciduous teeth (SHED) presented higher in vitro osteogenic differentiation potential (OD) as compared to MSCs derived from human adipose tissue (hASCs). Through microarray analysis and cell sorting, we have shown that IGF2 and CD105 expression levels contribute to these osteopontential cell differences, that is, higher IGF2 expression levels and lower CD105 expression levels were associated with the increased osteogenic potential of SHED as compared to hASCs. The molecular mechanisms associated with the diferent expression levels of IGF2 and CD105 in these cells were also investigated. Despite the advantages of adult MSCs they can exhibit drawbacks such as restricted self-renewal and limited cell amounts. Induced Pluripotent Stem Cells (iPSC) technology has emerged as an alternative cell source, as they provide more homogeneous cellular populations with prolonged self-renewal and higher plasticity. We verified that the OD of MSC-like iPSC differs from MSCs and it depends on the iPSCs originating cellular source. Comparative in vitro osteogenesis analysis showed higher osteogenic potential in MSC-like cells derived from iPS-SHED when compared with MSC-like cells from iPS-FIB and SHED. iPSCs can be also used as a tool to model genetic disorders. We have thus proposed to verify if it could be possible to in vitro model Treacher-Collins syndrome, a condition with deficient craniofacial bone development. We have compared the effects of pathogenic mutations in TCOF1 gene in cell proliferation, differentiation potential between MSCs, dermal fibroblasts, neural-crest like and MSC-like cells differentiated from iPSCs.TCS cells showed changes in cell properties anddysregulated expression of chondrogenesis markers during osteogenic and chondrogenic differentiation. In summary, the comparative analysis of stem cells of different sources allow us to identify markers that may facilitate osteogenesis and that it is possible to establish an in vitro model to Treacher-Collins syndrome<br>O uso de células-tronco trata-se de uma abordagem terapêutica promissora para a engenharia de tecidos, devido à sua capacidade na regeneração de tecidos, e para modelamento in vitro de distúrbios genéticos humanos, uma vez que fornece um abastecimento contínuo de células com potencial de diferenciação. Nosso estudo se propos a identificar moléculas e mecanismos que contribuem na otimização da osteogênese de células-tronco mesenquimais (MSCs). Para atingir nossos objetivos exploramos as diferenças no potencial osteogênico (PO) de MSCs de diferentes fontes. Observamos que MSCs de polpa de dente decíduo humano (SHED) apresentaram maior PO em comparação com as MSC derivadas de tecido adiposo humano (hASCs). Através de análise de microarray de expressão e cell sorting, demonstramos que os níveis de expressão de IGF2 e CD105 contribuem para as diferenças do PO, onde a maior expressão de IGF2 e menor expressão de CD105 estão associadas a maior PO em SHED quando comparado as hASCs. Também investigamos os mecanismos moleculares associados aos diferentes níveis de expressão de IGF2 E CD105 em ambas as fontes celulares. Apesar das vantagens, as MSCs podem apresentar pontos negativos como restrita auto-renovação e menor quantidade de células. Células-tronco pluripotentes induzidas (iPSC) surgem como uma fonte celular alternativa, proporcionando populações celulares homogêneas com auto-renovação prolongada e maior plasticidade. O PO de MSC-like iPSC difere de MSCs, e este potencial é dependente da fonte celular em que as iPSCs são obtidas. Análise comparativa de PO in vitro demonstrou maior osteogênse em células MSC-like derivadas de iPS-SHED quando comparada as células MSC-like de iPSCs-fibroblastos e SHED. iPSCs também podem ser utilizadas como ferramenta para investigar doenças genéticas humanas. Propomos a modelagem in vitro da síndrome de Treacher-Collins (TSC), doença que acomete as estruturas craniofaciais durante o desenvolvimento ósseo. Comparamos os efeitos de mutações patogênicas no gene TCOF1 na proliferação celular, potencial de diferenciação entre MSCs, fibroblastos dérmicos, neural-crest like e células MSC-like diferenciadas de iPSCs. Células de pacientes TCS exibiram alterações em propriedades celulares e na expressão de marcadores osteogênicos e condrogênicos. Em resumo, a análise comparativa de células-tronco de diferentes fontes permitiu a identificação de marcadores e mecanismos que podem facilitar a osteogênese e tambem demonstramos que é possível modelar in vitro a síndrome de Treacher-Collins

The aim of this study was to identify the stem/progenitor cell markers, by immunohistochemistry, in order to highlight the cortical neurogenesis niches during the different gestational ages. To this end, the following stem/progenitor cell markers have been utilized: Sox2, Pax6, Pax2, WT1, Nestin, Vimentin and Calretinin. The expression of these markers have been compared with the expression of those markers of mature neurons and glial markers including Neurofilament (NF), neuron specific enolase (NSE), Synaptophysin (Syn), Glial fibrillary acid protein (GFAP) and S100β. Evaluating both stem/progenitor and mature cell markers lead to the identification of multiple stages of differentiation of neuronal and glial progenitors during gestation, in order to better understand the development of human cerebellar cortex.

The documentation and the characterization of human stem/progenitor cells of the liver is interesting subject of the current scientific literature. Stem/progenitor cells are a heterogenus population characterized by a range of morphological and immunohistochemical features from bile duct cells to hepatocytes. They are typically characterized by the self-renewal ability able to differentiate into diverse lineage after injury or damage.. This study evaluated the immunohistochemical expression of different type of cytokeratins (CK7, CK14 and CK19) at different gestational ages in order to identify stem/progenitor in fetal human liver. Objectives: The aim of this study was to identify the stem/progenitor cell markers, by immunohistochemistry, in order to highlight the immunoreactivity of CK7, CK14 and CK19 in human liver progenitor cells at different gestational ages. Material and Methods: Liver samples were obtained from 14 fetal liver from 7 to 38 weeks of gestation. Liver samples were formalin-fixed routinely processed and stained with with H&E for histology. Section were also immunostained with the following antibodies: anti-CK7, anti-CK14 and anti-CK19. Results: Cytokeratin 7 (CK7): From 7 to 12 weeks of gestation, the positivity for CK7 is evident in the cytoplasm of progenitor cells of hepatocytes. From 15 weeks to 19 weeks, its immunoreactivity is absent. At about 27 weeks up to 38 weeks, we have a moderate positivity than before. The bile ducts in the first 7 weeks of gestation are absent. From 15 weeks onwards, we have a strong positivity of CK7 in ductal cells, remaining until late gestational age. The positivity of CK7 in the bile ducts is cytoplasmic and perinuclear. Cytokeratin 14 (CK14): From 7 weeks to 12 weeks, a cytoplasmic positivity, mainly perinuclear, is present in the cytoplasm of progenitor cells. From 15 weeks to 19 weeks, no immunoreactivity was found in hepatoblasts. From 27 weeks up to 38 weeks, there is a positive recovery. On the other hand, there is no positive effect during the development of the ductal system. Cytokeratin 19 (CK19): From 7 to 12 weeks of gestation, we have an intense cytoplasmic positivity homogeneously spread at the level of progenitor cells. At 15 weeks it is more light and focal and then negativize and start again from 27 weeks. Like CK7, also the CK19 is intensely expressed in the bile ducts from 15 weeks and then maintained until the 38th gestational age studied. Conclusion: Immunohistochemistry with monoclonal anti-cytokeratin antibodies has revelated previously the presence of cytokeratins in embryonic and early fetal hapatocytes. With the differentiation of bile ducts at about the 10th week, cytokeratin, particulary CK19, disappears from liver cells but remains in bile duct cells. This study shows that the immunoreactivity of the analyzed cytokeratins, in particular CK7 and CK19, is well evident from the first weeks of gestation and is maintained even in late age.

Emerging evidence has shown that the capacity of a tumor to grow and propagate is dependent on a small subset of cells, termed “cancer stem cells“. Conventional treatments, however, may miss the cancer stem cells, which have been shown in several tumor types to be more resistant to standard chemotherapeutic agents, because the cancer stem cell survives and regenerates the neoplasm. The aim of this study was to investigate the applicability of quantum dots in detection and eradication of cancer stem cells. The properties of stem cells were inestigated in breast, pancreatic, ovarian cancer and melanoma cells. The accumulation and distribution of quantum dots was assessed by the means of laser scanning confocal and fluorescence lifetime imaging microscopy. It was shown, that anti-CD44 and quantum dot conjugates are internalized selectively inside CD44+ cells, while anti-CD44 and organic dye FITC conjugates remained attached to cell membrane for at least 24 hours. Moreover, the complex of quantum dot (QD) and photosensitizer chlorin e6 (Ce6) accumulates inside cells and initiate cell death upon QD-directed exposure of 470 nm irradiation. These results show the capability of quantum dots not only to detect and internalize stem-like cells but also to carry the therapeutic agent inside cells without losing its activity.<br>Pastaruoju metu vis gausėja įrodymų apie vėžyje esančią nedidelę subpopuliaciją, pavadintą „vėžio kamieninėmis ląstelėmis“. Teigiama, jog būtent vėžio kamieninės ląstelės yra atsakingos už naviko formavimą, atsparumą taikomai terapijai bei metastazių formavimą. Šio darbo tyrimo tikslas – ištirti nanodarinių pritaikymo galimybes vėžinių kamieninių ląstelių aptikimui ir sunaikinimui. Darbe buvo ištirtos krūties, kasos, kiaušidžių vėžio ir melanomos ląstelių savybės, pagal literatūroje nurodytą vėžio kamieninių ląstelių aptikimo metodiką. Panaudojant lazerinę skenuojančią konfokalinę ir gyvavimo trukmių vaizdinimo mikroskopiją buvo įvertintas kvantinių taškų, kaip galimų diagnostiką ir terapiją atliekančių nanodarinių kaupimosi ir lokalizacijos ląstelėse tyrimai. Buvo parodyta, jog antikūnais prieš paviršiaus antigeną CD44 padengti kvantiniai taškai selektyviai pateko tik į CD44+ fenotipo ląstelių vidų. Anti-CD44-FITC konjugatai taip pat selektyviai žymėjo norimas ląsteles, tačiau net po 24 valandų nepateko į ląstelių vidų. Taip pat buvo parodyta, jog kvantinio taško (KT) ir sensibilizatoriaus chlorino e6 (Ce6) nanodarinys lokalizuojasi ląstelių viduje pūslelėse ir sukelia ląstelių žūtį tik toms ląstelėms, kurioms pritaikytas apjungtas KT-Ce6 komplekso ir 470 nm spinduliuotės poveikis. Tokie rezultatai parodo, jog galima kvantinį tašką ne tik selektyviai nukreipti į norimas ląsteles, bet taip pat ir įnešti į jų vidų, neprarandant terapinės molekulės aktyvumo.

Ovarian cancer is characterised by late diagnosis, relatively poor survival and high recurrence due to the development of multidrug resistance (MDR – resistance to various types of drug). Cancer stem-like cells (CSCs) are thought to contribute to drug resistance due to the over-expression of ABC transporters which efflux chemotherapeutic drugs. Frequently overexpressed ABC transporters include Multidrug resistance protein 1 (MRP1), P-glycoprotein (Pgp) and Breast cancer resistance protein (BCRP). No definitive stem cell marker panel has been determined for ovarian CSC detection; investigation of multiple putative stem cell markers (CD133, CD24, CD44 and Aldehyde dehydrogenase (ALDH)) may prove beneficial to the identification of these cells. All three ABC transporters are heterogeneously expressed in the ovarian cancer cell lines studied. Functional MRP1 and Pgp activity was identified utilising the Calcein-AM assay. Stem cell markers CD133 and ALDH were undetectable by western blot (WB). However small populations of ALDH expressing cells were detected using the ALDEFLUOR™ assay. Low-level CD44 was observed by WB, and confirmed by IF. RT-qPCR and microarray analysis confirmed the gene expression of all three ABC transporters and stem cell markers. A potential CSC population of high Hoechst effluxing cells, called a side population (SP) was identified. However, the viability of the SP was greatly reduced. Chemotherapeutic resistance to carboplatin, paclitaxel and doxorubicin was determined in ovarian cancer cell lines. The ovarian cancer cell line 1847 supports CSC and MDR theories, showing greater colony and spheroid formation and is most resistant to chemotherapeutics. Heterogeneous ABC transporter and stem cell marker expression was confirmed in primary samples. IHC analysis confirmed marker expression was not correlated with patient survival. Correlations were observed between MRP1 and CD44 and ALDH, CD44 and Pgp, BCRP and ALDH expression. Cells derived from patient ascites were also capable of forming colonies and spheroids, indicative of CSC properties.

Osteosarcoma (OS) is the most common bone malignancy often producing aggressive tumours in adolescents. OS aetiology is poorly understood, however, recent studies suggest OS cancers contain a small population of cancer stem cells (CSC) which initiate tumour growth. The cancer stem cell hypothesis describes cancers as a hierarchical population of heterogeneous cells. It has been proposed that CSC are at the base of this hierarchy and are responsible for the initiation, growth and spread of the tumour and pose a therapeutic challenge due to enhanced chemotherapy resistance. This project had three aims: identifying whether OS cell lines contain subpopulations of putative CSC, identifying if CSC contribute to chemotherapeutic resistance and to elucidate paracrine cell signals controlling OS tumour growth. Eight OS cell lines (143B, Cal72, G292, HOS, MG63, MNNG-HOS, U2OS and SaOS-2) along with the breast cancer cell line MCF7 have been analysed for the presence of sub-population of cells expressing putative CSC markers (aldehyde dehydrogenase and CD117). The intracellular enzyme aldehyde dehydrogenase (ALDH) and tyrosine kinase receptor CD117 were found to be heterogeneously expressed amongst the cell lines. All cell lines when plated at low density could recapitulate the colony hierarchies based on variation in colony morphology (holoclones, meroclones and paraclones), this was originally observed in carcinoma cell lines and is further putative evidence a CSC hierarchy exists within these cell lines. ALDH expressing cells were found to be confined to the holoclones (in cell lines with ALDH populations comprising less than 10 % of the total population) indicating that putative CSCs reside within this population. All OS cell lines also expressed mesenchymal markers (high vimentin and CD44 expression and low e-cadherin expression) suggesting they are a progenitor of mesenchymal stem cells. The expression of CD117 was found to negatively correlate with cisplatin chemotherapy resistance whereas ALDH inhibition using the specific antagonist diethylaminobenzalde sensitised different cells lines to opposing chemotherapeutics, suggesting a heterogenous response of OS ALDH cells to cytotoxic compounds. All OS cells lines, except 143B and HOS, were found to secrete a paracine growth factor which was capable of significantly enhancing their own growth. U2OS conditioned media was also able to enhance the growth of the breast cancer cell line MCF7 and a fibrosarcoma cell line (HT1080). Analysis of the cytokine expression profile of OS cell lines (HOS, MG63 and U2OS) demonstrated these cells secrete a broad range of cytokines associated with inflammation. The cytokine CCL-2 was identified as the putative OS paracrine growth factor as determined by the response to recombinant CCL-2, receptor antagonism and CCL-2 RNA interference. Genes with altered expression in response to CCL-2 were associated with transcription, suggesting that CCL-2 enhances proliferation through its downstream effect on transcription. Overall this thesis has contributed to the field of oncology by further defining the populations of putative CSC within a panel of OS cell lines. In addition the identification of a novel OS growth factor provides a possible adjuvant therapeutic target, which could aid in the reduction of OS proliferation.

The work reported in this thesis describes the characterisation of cell derived colonies from cancer cell lines and mechanisms of a key signalling pathway within these. PC3 cells were cultured at clonal density and resultant colony formation objectively described. Wnt signalling was investigated in colony types using a live [Ca2+]i assay. Exploratory work regarding ion channel gene and protein expression in 3 prostate cancer cell lines was performed. Preliminary investigation on the effects of MPRC (membrane potential regulating compounds) was performed using a cell proliferation assay. PC3 prostate cancer cells form distinct colonies with different morphologies. These different colony types can be characterised using quantitative parameters such as cell density and percentage cellularity, unlike previously employed visual only characteristics. PC3 cells form two types of colonies from a single cell. In this thesis these are termed holo/meroclones and paraclones. Cells within both colony types respond to Wnt activation, demonstrated by a response in free intracellular calcium [Ca2+]i . In addition, there is a suggestion of increased expression of, and translocation into the nucleus of the transcription factor co- activator β-catenin again in response to both Wnt 5A and Wnt 9B in both colony types. The characteristics of Wnt induced calcium release vary between different types of colonies. This is the first report of Wnt mediated [Ca2+]i release in response to both Wnt 5A and Wnt 9B in different PC3 colony types and the first report suggestive of β-catenin translocation following Wnt activation in colonies derived from stem-like cells.

Connor O’Hara July 29, 2019 Inhibition of Cancer Stem Cells by Glycosaminoglycan Mimetics In the United States cancer is the second leading cause of death, with colorectal cancer (CRC) being the third deadliest cancer and expected to cause over 51,000 fatalities in 2019 alone.1 The current standard of care for CRC depends largely on the staging, location, and presence of metastasis.2 As the tumor grows and invades nearby lymph tissue and blood vessels, CRC has the opportunity to invade not only nearby tissue but also metastasize into the liver and lung (most commonly).3 The 5-year survival rate for metastasized CRC is <15%, and standard of care chemotherapy regimens utilizing combination treatments only marginally improve survival.3-5 Additionally, patients who have gone into remission from late-stage CRC have a high risk of recurrence despite advances in treatment.6-7 The Cancer Stem-like Cell (CSC) paradigm has grown over the last 20 years to become a unifying hypothesis to support the growth and relapse of tumors previously regressed from chemotherapy (Figure 1).8 The paradigm emphasizes the heterogeneity of a tumor and its microenvironment, proposing that a small subset of cells in the tumor are the source of tumorigenesis with features akin to normal stem cells.9 The CSCs normally in a quiescent state survive this chemotherapy and “seed” tumor redevelopment.10 First observed in acute myeloid lymphoma models, CSCs have since been identified in various other cancers (to include CRC) by their cell surface antigens and unique properties characterizing them from normal cancer cells.11-12 These include tumor initiation, limitless self-renewal capacity to generate clonal daughter cells, as well as phenotypically diverse, mature, and highly differentiated progeny.13-14 Previously our lab has identified a novel molecule called G2.2 (Figure 2) from a unique library of sulfated compounds showing selective and potent inhibition of colorectal CSCs in-vitro.15 G2.2 is a mimetic of glycosaminoglycans (GAGs) and belongs to a class of molecules called non-saccharide GAG mimetics (NSGMs). Using a novel dual-screening platform, comparisons were made on the potency of G2.2 in bulk monolayer cells, primary 3D tumor spheroids of the same cell line, and subsequent generations of tumor spheroids. This work has shown in-vitro the fold-enhancement of CSCs when culturing as 3D tumor spheroids. Spheroid culture serves as a more accurate model for the physiological conditions of a tumor, as well as the functional importance of upregulating CSCs. Evaluation of G2.2 and other NSGMs was performed in only a few cell lines, developing a need to better understand the ability of G2.2 to inhibit spheroids from a more diverse panel of cancer cells to better understand G2.2’s mechanism. The last few decades have seen the advancement in fundamental biological and biochemical knowledge of tumor cell biology and genetics.16 CRC, in particular, has served as a useful preclinical model in recapitulating patient tumor heterogeneity in-vitro.17 Recent work has characterized the molecular phenotypes of CRC cell lines in a multi-omics analysis, stratifying them into 4 clinically robust and relevant consensus molecular subtypes (CMS).18-19 Our work was directed to screen a panel of cells from each of the molecular subtypes and characterize the action of G2.2 and 2nd generation lipid-modified analogs, synthesized to improve the pharmacokinetic properties of the parent compound. Four NSGMs, namely G2.2, G2C, G5C, and G8C (Figure 2) were studied for their ability to inhibit the growth of primary spheroids across a phenotypically diverse panel. Compound HT-29 IC50 (μM) Panel Average IC50 (μM) G2.2 28 ± 1 185 ± 55 G2C 5 ± 2 16 ± 15 G5C 8 ± 2 63 ± 19 G8C 0.7 ± 0.2 6 ± 3 Primary spheroid inhibition assays were performed comparing the potency of new NSGMs to G2.2. Fifteen cell lines were evaluated in a panel of colorectal adenocarcinoma cell lines with several cell lines representing each CMS. Primary spheroid inhibition assays revealed 3 distinct response with regard to G2.2’s ability to inhibit spheroid growth. Cells from CMS 3 and 4, which display poor clinical prognosis, metabolic dysregulation, and enhanced activation of CSC pathways, showed the most sensitivity to G2.2 (mean IC50 = 89 ± 55 μM). Mesenchymal CMS 4 cell lines were over 3-fold more sensitive to treatment with G2.2 when compared to CMS 1 cell lines. Resistant cell lines were composed entirely of CMS 1 and 2 (mean IC50 = 267 ± 105 μM). In contrast, all lipid-modified analogs showed greater potency than the parent NSGM in almost every CRC cell line. Of the three analogs, G8C showed the greatest potency with a mean IC50 of less than 15 μM. Of the CRC spheroids studied, HT-29 (CMS 3) was most sensitive to G8C (IC50 = 0.73 μM). To evaluate the selectivity of NSGMs for CSC spheroid inhibition, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) cytotoxicity assays were performed on monolayer cell culture, and the fold-selectivity of NSGM for spheroids was analyzed. Data shows that NSGMs preferentially target CSC-rich spheroids compared with monolayer cellular growth, with G2.2 having over 7-fold selectivity for spheroid conditions. This fold selectivity was enhanced in CMS 3/4, supporting the idea that G2.2 targets a mesenchymal and stem-like phenotype. To further validate this selectivity, limiting dilution assays were performed across the panel to determine the tumor-initiating capacity of each cell line. Cell lines which showed a sensitive response to G2.2 were over 2-fold more likely to develop into spheroids, validating the previous hypothesis. Further characterization was performed analyzing the changes G2.2 induced on CSC markers, as well as the basal expression of a unique pair of cancer cells. Western blots showed a reduction in self-renewal marker across all CMS after treatment with G2.2, and that cell lines sensitive to G2.2-treatment overexpress mesenchymal and stem-like markers. G2.2-resistant cell lines show an epithelial phenotype, lacking this expression. The positive results observed in these studies enhance the understanding of G2.2 and analogs, and further evaluation with additional cell lines of various tissues would improve the knowledge thus far gained. However, all experiments described take valuable time to perform and analyze. Thus, there became a need to develop a high-throughput screening (HTS) platform for our assays that standardized analysis and enhanced productivity. Initial development of the method for this assay are underway, and recent evidence from these evaluations of breast cancer spheroids suggests that G2.2 and analogs may be tissue-specific compounds for the treatment of cancer. Future work entails refining the application of this method for evaluation of the NCI-60 (National Cancer Institute) tumor cell panel. Overall, these results make several suggestions concerning the NSGMs evaluated against the panel. First, G2.2 selectively targets CSCs with limited toxicity to monolayer cells of the same cell line. Further, G2.2 has the greatest potency with CMS 3/4, whose mesenchymal phenotypes are associated with poor clinical prognosis and enrichment of CSCs. Supporting evidence include that sensitive cell lines are highly tumorigenic and show enhanced expression of mesenchymal/CSC markers compared to resistant cell lines. Lipid-modification of G2.2 enhances in-vitro potency against spheroid growth, with nM potency reached in the most sensitive cell lines. Evidence in the development of a HTS platform also suggests these NSGMs show tissue specificity to cancers of the intestine. Further work characterizing the mechanism of NSGMs in a broader multi-tissue panel will enhance our understanding of the compounds as a potential therapy to dramatically improve patient survival through specific targeting of tumorigenesis. References 1. Colorectal Cancer Facts & Figures 2017-2019. American Cancer Society 2017. 2. Compton, C. C.; Byrd, D. R.; Garcia-Aguilar, J.; Kurtzman, S. H.; Olawaiye, A.; Washington, M. K. Colon and rectum. In AJCC Cancer Staging Atlas, 2nd ed.; Ed. Springer Science: New York, 2012; pp 185–201. 3. Van Cutsem, E.; Cervantes, A.; Adam, R.; Sobrero, A.; Van Krieken, J. H.; Aderka, D.; Aranda Aguilar, E.; Bardelli, A.; Benson, A.; Bodoky, G.; et al. ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Ann. Oncol. 2016, 27, 1386–422. 4. Siegel, R. L.; Miller, K. D.; Fedewa, S. A.; Ahnen, D. J.; Meester, R. G. S.; Barzi, A.; Jemal, A. Colorectal cancer statistics, 2017. CA Cancer J. Clin. 2017, 67, 177–193. 5. Moriarity, A.; O'Sullivan, J.; Kennedy, J.; Mehigan, B.; McCormick, P. Current targeted therapies in the treatment of advanced colorectal cancer: a review. Ther. Adv. Med. Oncol. 2016, 8, 276–293. 6. Seidel, J.; Farber, E.; Baumbach, R.; Cordruwisch, W.; Bohmler, U.; Feyerabend, B.; Faiss, S. Complication and local recurrence rate after endoscopic resection of large high-risk colorectal adenomas of >/=3 cm in size. Int. J. Colorectal Dis. 2016, 31, 603–611. 7. Pugh, S. A.; Shinkins, B.; Fuller, A.; Mellor, J.; Mant, D.; Primrose, J. N. Site and stage of colorectal cancer influence the likelihood and distribution of disease recurrence and postrecurrence survival: data from the FACS randomized controlled trial. Ann. Surg. 2016, 263, 1143–1147. 8. Batlle, E.; Clevers, H. Cancer stem cells revisited. Nat. Med. 2017, 23, 1124–1134. 9. Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 2011, 144, 646–674. 10. Tirino, V.; Desiderio, V.; Paino, F.; De Rosa, A.; Papaccio, F.; La Noce, M.; Laino, L.; De Francesco, F.; Papaccio, G. Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization. FASEB J. 2013, 27, 13–24. 11. Bonnet, D.; Dick, J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 1997, 3, 730–737. 12. Desai, A.; Yan, Y.; Gerson, S. L. Concise reviews: cancer stem cell targeted therapies: toward clinical success. Stem Cells Transl. Med. 2019, 8, 75–81. 13. Munro, M. J.; Wickremesekera, S. K.; Peng, L.; Tan, S. T.; Itinteang, T. Cancer stem cells in colorectal cancer: a review. J. Clin. Pathol. 2018, 71, 110–116. 14. Zhou, Y.; Xia, L.; Wang, H.; Oyang, L.; Su, M.; Liu, Q.; Lin, J.; Tan, S.; Tian, Y.; Liao, Q.; Cao, D. Cancer stem cells in progression of colorectal cancer. Oncotarget 2018, 9, 33403–33415. 15. Patel, N. J.; Karuturi, R.; Al-Horani, R. A.; Baranwal, S.; Patel, J.; Desai, U. R.; Patel, B. B. Synthetic, non-saccharide, glycosaminoglycan mimetics selectively target colon cancer stem cells. ACS Chem. Biol. 2014, 9, 1826–1833. 16. Punt, C. J.; Koopman, M.; Vermeulen, L. From tumour heterogeneity to advances in precision treatment of colorectal cancer. Nat. Rev. Clin. Oncol. 2017, 14, 235–246. 17. Mouradov, D.; Sloggett, C.; Jorissen, R. N.; Love, C. G.; Li, S.; Burgess, A. W.; Arango, D.; Strausberg, R. L.; Buchanan, D.; Wormald, S.; et al. Colorectal cancer cell lines are representative models of the main molecular subtypes of primary cancer. Cancer Res. 2014, 74, 3238–3247. 18. Guinney, J.; Dienstmann, R.; Wang, X.; de Reynies, A.; Schlicker, A.; Soneson, C.; Marisa, L.; Roepman, P.; Nyamundanda, G.; Angelino, P.; et al. The consensus molecular subtypes of colorectal cancer. Nat. Med. 2015, 21, 1350–1356. 19. Berg, K. C. G.; Eide, P. W.; Eilertsen, I. A.; Johannessen, B.; Bruun, J.; Danielsen, S. A.; Bjornslett, M.; Meza-Zepeda, L. A.; Eknaes, M.; Lind, G. E.; et al. Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies. Mol. Cancer 2017, 16, 116–132.

Cancer Stem cells (CSCs), recently identified in the majority of solid tumors, are thought to drive tumor growth, giving rise to a cascade of differentiated cells composing the bulk of the tumor. Tumor relapse that most often follows treatment with anti-proliferative and cytotoxic drugs would be explained by selective resistance of CSC to these agents. Colon cancer stem cells (cCSCs), first isolated in the host laboratory from surgical specimens, can be grown in vitro as clusters called tumor spheres that maintain an undifferentiated state and are able, upon injection in immunodeficient mice, to generate a xenograft identical to the parental tumor, in terms of both antigen expression and histological tissue organization. Because of all these features cCSCs may represent predictive tools for patient s therapeutic response. Cetuximab (Erbitux), currently in use for metastatic colorectal cancer, is a recombinant chimeric human:murine immunoglobulin IgG1 that binds to EGFR displacing its natural ligands. Cetuximab also induces receptor internalization and degradation. Mutations in signaling pathway mediators acting downstream of EGFR, including KRAS, BRAF, NRAS, or PIK3CA are believed to determine resistance to the drug. In particular, KRAS-mutated patients are currently excluded from treatment. In order to verify whether Cetuximab treatment affects the stem cell compartment within tumors, in this study I analyzed its effect in a panel of cCSCs generated by individual patients, both in vitro and in xenografts. The data show that the effect of Cetuximab on individual cCSCs reflects the known clinical data on individual tumor mutations in the EGFR signaling pathway molecules. The study therefore confirms that panels of cCSCs generated by individual patients represent good predictive tools for the preclinical screening of pathway-oriented, cancer stem cell-directed therapeutics. Most importantly, the analysis of stem cell content in Cetuximab-treated xenografts by cytofluorimetry, agarose assay, and serial re-transplantation into secondary hosts clearly demonstrate that Cetuximab, differently than the classical chemotherapeutics currently in use for colon cancer, is able to effectively hit cCSC populations included in the tumors.

The incidence of Human papillomavirus (HPV) induced oropharyngeal squamous cell carcinoma (OSCC) is rising. HPV-positive OSCC is associated with a favourable prognosis, the mechanism is not fully understood and could be related to intrinsic radiosensitivity of the HPV-positive OSCC. The aim of this candidature was to study the influence of tumour microenvironment on radiosensitivity by examine HIF-1α in OSCC and by using in vitro 3D tumour models. The main finding of the HIF-1α study was the HPV status remains the strongest prognostic factor in OSCC. Hypoxia might be an important factor in HPV-negative cancer. In consistent with clinical observations, in vitro studies revealed HPV-positive OSCC cells were more sensitive to radiation than were HPV-negative OSCC cells. Radiosensitivity of HPV-positive OSCC was related to G1/S cell cycle regulation, increased cyclin D1 expression and apoptosis; associated with p16/CDK/Rb pathways. HPV-negative OSCC cells displayed a radiation-induced G2 arrest indicating the activation of DNA repair mechanism. Radiation induced cyclin D1 expression indicated that responsible for HPV-positive OSCC radiosensitivity. The results from this candidature emphasize the important roles of HPV and microenvironment on cancer cell response to radiation. The results provided a biological basis for using different strategies when treating HPV-positive and HPV-negative OSCC.

Colorectal cancer (CRC) is the third most common form of cancer and the second cause of cancer-related death in the Western world. Despite the emergence of new targeted agents and the use of various therapeutic combinations, none of the treatments options available is curative in patients with advanced cancer. In the last decade, several studies have shown that a small population of “stem cell like” cells, with the capacity for self-renewal, multipotency and high tumorigenic potential, could be isolated from tumors with different histological origin, including human CRC. These peculiar stem cells are believed to be responsible for tumor initiation, progression and resistance to therapeutic agents. Therefore, treatments that are design to eradicate tumor masses should be also targeted to CSCs. Recently in our laboratory cancer stem cells (CSCs) lines and their fetal bovine serum (FBS)-cultured non-CSC pair lines were in vitro isolated from surgical specimens of patients with primary CRC cancer. In this study we performed an immunobiological characterization of CRC CSCs and investigated whether these cells can represent suitable targets for immunotherapy protocols. The phenotype and functional characterization of these cell lines was examined by IF and cytofluorimetric analysis. Firstly the expression levels of CRC cancer and/or stem cell-associated molecules such as: Ep-CAM, CEA, HCAM, CD44, Aldefluor, CD133, SOX-2, Nanog and Oct-4 was assessed. Notably, the tumorigenic potency of CSCs was higher than that of FBS cells as shown by the serial dilutions and serial transplantations cell injection in immunodeficient mice. Moreover the xenografts generated by CRC CSC showed large areas of necrosis than tumor tissues generated by FBS cells and better matched the phenotype of the original tumor, as revealed by an IHC analysis of CRC and CSC-associated molecules. In addition, the expression of MHC-I and MHC-II molecules, NKG2D ligands, immune regulatory molecules, antigen-processing machinery (APM) and tumor associated antigens (TAA) was examined. Both CRC CSC and FBS lines were found weakly positive for MHC-I and negative for MHC-II, while NKG2D ligands were commonly detected in both cells types. The APM was mainly defective in CRC CSC versus FBS cell lines. We also investigated whether the expression of immune-regulatory molecules (CTLA-4, PD-1, B7-H3) can affect the immunogenicity of these cells. All CSCs and FBS cell lines were homogeneously positive for CTLA-4, PD-1 and, although at higher levels for PD-L1 and B7-H3. All these molecules acts as negative modulator of T-cells response because of the inhibition of the vital ‘second signal’ required for optimal T cell recognition and activation. Interestingly, IL-4 was expressed by all the cell lines both in the cytoplasm and in the membrane while IL-4R was weakly detectable on the membrane of CSC and was absent in FBS lines counterparts. These data may confirm the possibility of an autocrine production by CRC CSC and also FBS line of IL-4, that may thus protect themselves by apoptosis as recently described by Stassi’s group (Todaro M et al, 2007), this citockine may also induce an immunomodulatory role for T-cells mediated responses against CRC. Unexpectedly FBS tumor lines released in the supernatants high levels of prostaglandin-E2 (PGE2) that, on the contrary was weakly or absent in the supernatant of CSCs. In addition, T lymphocytes isolated from the peripheral blood of CRC patients were stimulated in vitro with autologous CSCs or FBS, in order to assess of anti-CRC reactivity. In patient #1076 we could isolate mostly TH1-mediated responses (detected by INF-γ release), showing weak reactivity against CSCs. On the contrary a more efficiently INF-γ release was detectable when T-lymphocytes were stimulated with FBS tumor cells. In a second patient (#1247), a MHC-independent recognition of CSCs was observed, suggesting an NK-mediated response. This result was also confirmed by the phenotype analysis of these lymphocytes that revealed the presence of 20-30% NK cells (CD3-CD16*CD56+).These findings correlate with the defective expression of molecules involved in antigen presenting and processing machinery observed in CSCs. Taken together our data we can conclude that we have isolate from CRC tissues cells with stemness properties, thus showing a lower immunogenicity compared with the FBS counterpart. T cell responses could be obtained in CRC patients directed against CSC and FBS cells, though with higher reactivity for FBS tumor cells, while in another patient NK-mediated responses could be isolated These findings may be useful for the identification of new agents that can efficiently rescue the immunogenicity of CSCs in order to be targeted by T cell-mediated immune response.

Cancer Stem Cells (CSCs) are a clinically relevant population at the apex of the inner hierarchical organization of many tumors. It was previously demonstrated by our group that loss of the p53 tumor suppressor leads to an increase in the mammary stem cell (MaSC) and breast CSC content, due to a switch of the mode of division from mainly asymmetric to symmetric. However, which of the many pathways instructed by p53 is directly involved in the execution of this biological phenotype remains to be determined. Following a candidate gene approach, we investigated Myc as the putative key downstream effector of p53 in breast SCs and CSCs. The Myc oncogene is very often altered in cancer and has been clinically associated with poor differentiation and aggressiveness in breast cancer. We have found that Myc endogenous expression is de- regulated in our ErbB2 model of breast tumorigenesis, upon attenuation of p53 signaling. We also observed that de-regulated Myc extends the lifespan and proliferative potential of wild type mammospheres. This occurs by two distinct but cooperative mechanisms: the increase in the frequency of symmetric divisions of MaSCs and the reprogramming of progenitor cells. Importantly, in the ErbB2 model, de-regulated Myc levels are critical and sufficient to sustain the unlimited self-renewal of CSCs, independently of p53. Of note, the above described phenotype is characterized by the over expression of a mitotic gene signature which is dictated by the identified p53-Myc axis. Taken together these results demonstrate that the loss of a tight control on Myc levels, which derives from the loss of p53 functionality, is responsible for the expansion of the SC and CSC pool by regulating modality of SC division and reprogramming of mammary progenitors. Finally, our data suggest that the p53-Myc axis exerts a putative tumor suppressor function in SCs through the coordinated regulation of a set of mitotic genes.

The Cancer Stem Cell (CSC) theory suggests that cancers arise from and are maintained by a subpopulation of cancer cells with stem cell properties. Molecular chaperones are key components of cellular regulation. The overexpression of chaperones has become synonymous with cancer cells with chaperones being recognized as bona fide anti-cancer drug targets. Although chaperone activity has been characterized in cancer cells, very little is known about the cellular functions of chaperones in cancer stem cells. We set out to compare the expression of selected molecular chaperones in non-stem cancer cell and cancer stem cell enriched populations isolated from breast cancer lines, in order to identify chaperones differentially expressed between the two populations for further biological characterization. In order to isolate breast cancer stem cells from the MCF-7 and MDA-MB-231 breast cancer cell lines, three cancer stem cell isolation and identification techniques were utilized based on (1) cell surface marker expression (CD44+/CD24- and CD44+/CD24-/EpCAM+ phenotypes), (2) aldehyde dehydrogenase enzyme activity (ALDHHi) and (3) ability to grow in anchorage-independent conditions. The MDA-MB-231 and MCF-7 breast cancer cell lines displayed CD44+/CD24- cell populations with the MCF-7 cell line additionally displaying a large CD44+/CD24-/EpCAM+ population. Although both cell lines showed similar ALDHHi populations, they differed substantially with respect to anchorage-independent growth. MCF-7 cells were able to form anchorage-independent colonies while the MDA-MB-231 cell line was not. Anchorage-independent MCF-7 cells showed enrichment in CD44+/CD24- and CD44+/CD24-/EpCAM+ cells compared to adherent MCF-7 cells, and were selected for gene expression studies. Gene expression studies identified 22 genes as being down-regulated at the mRNA level in the anchorage-independent MCF-7 cells, while only 2 genes (BAG1 and DNAJC12) were up-regulated. The down-regulation of selected chaperones in anchorage independent MCF-7 cells was confirmed at the protein level for selected chaperones, including DNAJB6, a type II DNAJ protein shown to be involved in the regulation of Wnt signaling. In order to characterize the effect of DNAJB6 expression on BCSCs we developed a pCMV mammalian expression plasmid for both DNAJB6 isoforms (DNAJB6L and DNAJB6S). We successfully constructed mutants of the conserved histidine-proline-aspartic acid (HPD) motif of the J domain of DNAJB6S and DNAJB6L. These constructs will allow the analysis of the role of DNAJB6 in cancer stem cell function. To the best of our knowledge, this is the first report to focus on the comparative expression of molecular chaperones in normal and cancer stem cell enriched breast cancer populations.

The development of the human cerebral cortex represents a delicate moment of embryogenesis. The ventricular zone (VZ) and the subventricular zone (SVZ) are considered the “stem/progenitor cells niches” of the developing cerebral cortex. As the majority of studies of brain development have been focused mainly on animal models, this study was focused on normal human cerebral cortex development, in particular on the stem/progenitor cells markers that may play a key role during human corticogenesis. To this end, samples from cerebral cortex were obtained from 20 human fetuses from 9 up to 40 weeks of gestation. Each sample was formalin-fixed, paraffin embedded and immunostained with several markers including WT1, Sox2, Pax6, Vimentin, Nestin, Sox11, Pax2, NF, NSE, Synaptophysin, GFAP and S100B. Five important markers of radial glial progenitor cells were evidenced during the first half of gestation: Sox2, Pax6, Vimentin, Nestin and WT1. Vimentin, Nestin and WT1 were expressed in radial glia fibers which represent the most important guide for the radial migration of newborn neurons. Instead, Pax6 immunoreactivity was detected in the VZ and SVZ, being express in radial glia cell bodies. Sox2 was expressed by the stem/progenitor cells of the VZ and SVZ including radial glia and intermediate progenitor cells, and by migrating newborn neurons. Pax2 and Sox11 expression were detected in the progenitor cells of VZ and SVZ, and in the migrating newborn neurons. Immunoreactivity for S100B, GFAP, NF and Synaptophysin, was mild or totally absent in the first half of gestation. These data reflect the lack of maturation of glial and neuronal cells in the early phases of human corticogenesis. Future studies are needed to detect differences in the mRNA expression patterns of these markers in order to better evaluate their role in cell proliferation and differentiation during human intrauterine development.<br>The development of the human cerebral cortex represents a delicate moment of embryogenesis. The ventricular zone (VZ) and the subventricular zone (SVZ) are considered the “stem/progenitor cells niches” of the developing cerebral cortex. As the majority of studies of brain development have been focused mainly on animal models, this study was focused on normal human cerebral cortex development, in particular on the stem/progenitor cells markers that may play a key role during human corticogenesis. To this end, samples from cerebral cortex were obtained from 20 human fetuses from 9 up to 40 weeks of gestation. Each sample was formalin-fixed, paraffin embedded and immunostained with several markers including WT1, Sox2, Pax6, Vimentin, Nestin, Sox11, Pax2, NF, NSE, Synaptophysin, GFAP and S100B. Five important markers of radial glial progenitor cells were evidenced during the first half of gestation: Sox2, Pax6, Vimentin, Nestin and WT1. Vimentin, Nestin and WT1 were expressed in radial glia fibers which represent the most important guide for the radial migration of newborn neurons. Instead, Pax6 immunoreactivity was detected in the VZ and SVZ, being express in radial glia cell bodies. Sox2 was expressed by the stem/progenitor cells of the VZ and SVZ including radial glia and intermediate progenitor cells, and by migrating newborn neurons. Pax2 and Sox11 expression were detected in the progenitor cells of VZ and SVZ, and in the migrating newborn neurons. Immunoreactivity for S100B, GFAP, NF and Synaptophysin, was mild or totally absent in the first half of gestation. These data reflect the lack of maturation of glial and neuronal cells in the early phases of human corticogenesis. Future studies are needed to detect differences in the mRNA expression patterns of these markers in order to better evaluate their role in cell proliferation and differentiation during human intrauterine development.

Li Ka Shing Prizes for best PhD theses in the Faculties of Dentistry, Engineering, Medicine and Science, 2006-2007<br>published_or_final_version<br>abstract<br>Pathology<br>Doctoral<br>Doctor of Philosophy

Tumor heterogeneity has long been observed and recognized as a challenge to cancer therapy. The cancer stem cell (CSC) model is one of the hypotheses proposed to explain such a phenomenon. A specific cancer stem cell marker has not been determined for non-small cell lung cancers (NSCLC), preventing the definitive evaluation of whether the biology of NSCLC is governed by the CSC model. This study aimed to analyze the expression of candidate CSC markers and using the identified putative marker, to isolate CSC and determine the applicability of the CSC model in NSCLC. The expression or activities of four putative stem cell markers, CD24, CD44, CD133 and aldehyde dehydrogenase 1 (ALDH1) were measured by flow cytometry in eight NSCLC cell lines before and after chemotherapy for 24 hours. Markers with increased expression after treatment were considered potential CSC markers and used for isolating tumor cell subpopulations from the untreated cell lines by fluorescence-activated cell sorting (FACS). Confirmatory analyses using widely acceptable methodology were performed to test for CSC properties in the marker+ and marker- subpopulations. Isolated subpopulations were further characterized by functional and phenotypic studies. Flow cytometry showed amongst the 4 markers, only ALDH1 expression was significantly enhanced by chemotherapeutic treatment, suggesting ALDH1 could be a CSC marker. Untreated ALDH1+ cells formed significantly more and larger cell spheres in non-adherent, serum-free conditions than ALDH1- cells. Likewise, higher in vitro tumorigenic ability was observed in ALDH1+ subset using colony formation assay. Furthermore, a higher resistance to cytotoxic drugs was observed in ALDH1+ compared to ALDH1- cells. In vivo studies also showed ALDH1+ cells showed higher tumorigenicity than ALDH1- cells; as few as 2,500 ALDH1+ cells formed tumor in SCID mice which were serially transplantable to 2nd and 3rd recipients, while no tumor was formed from ALDH- cells with even ten times the number of cells. Also, expression analysis revealed higher expression of the pluripotency genes, OCT4, NANOG, BMI1 and SOX9, in ALDH1+ cells. In view of previous studies reporting CD44 as a CSC marker in lung cancer, double marker selection of putative CSC was performed to compare ALDH1+CD44+ and ALDH1-CD44+ subpopulations. Results of the spheroid body formation assay and cisplatin treatment experiments revealed the ALDH1+CD44+ subpopulation possessed higher self-renewal ability and chemo-resistance. Cell migration and invasion assays showed differences between the ALDH1+CD44+ and ALDH1- CD44+ subpopulations. The significance of these observations require further investigation. In conclusion, the result showed that ALDH1 could be a marker for NSCLC stem cells as evidenced by enhanced self-renewal and differentiation abilities in ALDH1+ subpopulation. Furthermore, this study observed the presence of at least two potential stem cell subpopulations in NSCLC cells with differential selfrenewal, chemotherapy resistance and cell mobility properties. Further investigations are required to validate these observations and to investigate the underlying mechanisms. Better understanding of these issues would help to solve the challenges brought by tumor heterogeneity in lung cancer therapy.<br>published_or_final_version<br>Pathology<br>Master<br>Master of Philosophy

Oshima N, Yamada Y, Nagayama S, Kawada K, Hasegawa S, et al. (2014) Induction of Cancer Stem Cell Properties in Colon Cancer Cells by Defined Factors. PLoS ONE 9(7): e101735. doi:10.1371/journal.pone.0101735<br>Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(医学)<br>甲第18547号<br>医博第3940号<br>新制||医||1006(附属図書館)<br>31447<br>京都大学大学院医学研究科医学専攻<br>(主査)教授 千葉 勉, 教授 野田 亮, 教授 武藤 学<br>学位規則第4条第1項該当