Photodynamic therapy in cancer stem cells - state of the art.

Despite significant efforts to control cancer progression and to improve oncology treatment outcomes, recurrence and tumor resistance are frequently observed in cancer patients. These problems are partly related to the presence of cancer stem cells (CSCs). Photodynamic therapy (PDT) has been developed as a therapeutic approach for solid tumors; however, it remains unclear how this therapy can affect CSCs. In this review, we focus on the effects of PDT on CSCs and the possible changes in the CSC population after PDT exposure. Tumor response to PDT varies according to the photosensitizer and light parameters employed, but most studies have reported the successful elimination of CSCs after PDT. However, some studies have reported that CSCs were more resistant to PDT than non-CSCs due to the increased efflux of photosensitizer molecules and the action of autophagy. Additionally, using different PDT approaches to target the CSCs resulted in increased sensitivity, reduction of sphere formation, invasiveness, stem cell phenotype, and improved response to chemotherapy. Lastly, although mainly limited to in vitro studies, PDT, combined with targeted therapies and/or chemotherapy, could successfully target CSCs in different solid tumors and promote the reduction of stemness, suggesting a promising therapeutic approach requiring evaluation in robust pre-clinical studies.

The Role of lncRNAs in the Stem Phenotype of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma is one of the deadliest tumors. This neoplasia is characterized by an important cellular and phenotypic heterogeneity. In particular, it has been shown that at least two subtypes can be found: basal-like, which presents stem-like properties, and classical. Cancer stem cells have been isolated and characterized from these tumors, showing their dependance on general and tissue-specific stem transcription factors and signaling pathways. Nevertheless, little is known about their tissue microenvironment and cell non-autonomous regulators, such as long-non-coding RNAs. (lncRNAs). In this review, we summarize the current knowledge about the positive and negative effects of lncRNAs in the stemness phenotype of pancreatic ductal adenocarcinoma cancer (PDAC).

Childhood Acute Leukemias in Developing Nations: Successes and Challenges.

PURPOSE OF REVIEW: Acute leukemias represent a tremendous threat to public health around the globe and the main cause of death due to disease in scholar age children from developing nations. Here, we review their current status in Mexico, as a paradigm of study, and the major challenges to control systemic diseases like childhood cancer. RECENT FINDINGS: A unique molecular epidemiology, late/low precision diagnosis, limited access to treatment, toxicity associated with therapy, continuous exposure to environmental risk factors, and the high frequency of early relapses are some of the factors cooperating to low rates of survival in low-to-medium-income countries. Deliberative dialogues and exhaustive programs have emerged as promising means of advancing evidence-informed policy, by providing a structured forum for key stakeholders to integrate scientific and pragmatic knowledge about complex health concerns. A system-wide strategy based on the comprehensive leukemia identity is essential for a meaningful decline in early childhood mortality.

Role of glioblastoma stem cells in cancer therapeutic resistance: a perspective on antineoplastic agents from natural sources and chemical derivatives.

Glioblastoma (GBM) is the highest-grade form of glioma, as well as one of the most aggressive types of cancer, exhibiting rapid cellular growth and highly invasive behavior. Despite significant advances in diagnosis and therapy in recent decades, the outcomes for high-grade gliomas (WHO grades III-IV) remain unfavorable, with a median overall survival time of 15-18 months. The concept of cancer stem cells (CSCs) has emerged and provided new insight into GBM resistance and management. CSCs can self-renew and initiate tumor growth and are also responsible for tumor cell heterogeneity and the induction of systemic immunosuppression. The idea that GBM resistance could be dependent on innate differences in the sensitivity of clonogenic glial stem cells (GSCs) to chemotherapeutic drugs/radiation prompted the scientific community to rethink the understanding of GBM growth and therapies directed at eliminating these cells or modulating their stemness. This review aims to describe major intrinsic and extrinsic mechanisms that mediate chemoradioresistant GSCs and therapies based on antineoplastic agents from natural sources, derivatives, and synthetics used alone or in synergistic combination with conventional treatment. We will also address ongoing clinical trials focused on these promising targets. Although the development of effective therapy for GBM remains a major challenge in molecular oncology, GSC knowledge can offer new directions for a promising future.

Identification of very small cancer stem cells expressing hallmarks of pluripotency in B16F10 melanoma cells and their reoccurrence in B16F10-derived clones

Melanoma is characterized by high heterogeneity and plasticity, most likely due to the presence of mutated melanocyte stem cells or immature progenitor cells in the skin that serves as precursors to melanoma. In the present study, for the first time, we identified rare cells in the murine melanoma B16F10, and human A2058 and SK-MEL-28?cell lines that express pluripotency markers, including Oct4, Nanog, Sox2 and a marker of melanoma cancer cells (ALDH1/2). These cells are very small with round morphology and they grow onto melanoma cells, thereby demonstrating feeder layer dependence similar to that of other pluripotent cells. These cells underwent self-renewal, symmetric and asymmetric division. We called these cells murine very small cancer stem cells (VSCSC). VSCSC were also found in B16F10-derived clones after 3–5 consecutive passages, where they occur as single cells or as small colonies, nevertheless, always using melanoma cells as feeders. These cells formed melanospheres enriched with Oct4-and ALDH1/2-positive cells. We also evaluated the possible effect of VSCSC that presented in the parental cell line (B16F10) and in clones based on their functional characteristics. We found that VCSCS present in the B16F10?cell line reappearing in their clones were required for continuous tumor growth and were responsible for melanoma cell heterogeneity and plasticity rather than directly affecting functional characteristics of melanoma cells. Our data, together with those of previous reports suggested the existence of melanoma-competent melanocyte stem cells, which corroborate the hypothesis of the existence of tumor-initiating cells and cancer stem cell hierarchies, at least in melanoma

Explorando a quinase IKK como um alvo terapêutico para células iniciadoras de tumor pulmonares induzidas pelo oncogene KRAS / Exploring IKKb kinase as a therapeutic target for KRAS-driven lung tumour-initiating cells

As alterações genéticas mais frequentes em câncer de pulmão são mutações pontuais que ativam o oncogene KRAS. Embora estas mutações estejam causalmente relacionadas à oncogênese, até hoje diferentes abordagens para inibir as proteínas RAS diretamente não obtiveram sucesso. Portanto, para que melhores alvos terapêuticos para o câncer de pulmão se tornem disponíveis é necessário identificar os mecanismos moleculares ativados por KRAS que estão diretamente envolvidos com a aquisição de propriedades malignas importantes, como o desenvolvimento e a manutenção de um fenótipo tronco-tumoral pelas células iniciadoras de tumor (CITs). CITs, também conhecidas como células tronco-tumorais, são definidas como uma subpopulação de células tumorais capazes de se autorrenovar, iniciar a formação de tumores e sustentar o crescimento tumoral. O desenvolvimento de estratégias terapêuticas dirigidas a estas células é imprescindível para melhorar a eficácia da terapia antitumoral. Uma vez que KRAS está associada a manutenção de um fenótipo tronco-tumoral e ativa o fator de transcrição NF-kB através da quinase IKKß para promover a tumorigênese pulmonar, nós hipotetizamos que a quinase IKKß contribui para o fenótipo tronco-tumoral induzido por KRAS em câncer de pulmão. Nós utilizamos ensaios de formação de tumoresferas para enriquecer e avaliar a função de CITs das linhagens pulmonares positivas para KRAS A549 e H358. As células A549 e H358 formaram tumoresferas em cultura de baixa aderência e, quando comparadas às células derivadas da cultura aderente, as células oriundas da cultura de tumoresferas apresentaram maior crescimento clonogênico, maior expressão de genes associados ao fenótipo tronco por qPCR e maior atividade da quinase IKKß. A inibição da atividade de IKKß através de um inibidor farmacológico altamente específico (Composto A) diminuiu levemente a proliferação de células A549 e H358, sem resultar em morte celular significativa. Entretanto, a inibição da atividade ou da expressão de IKKß por interferência de RNA reduziu a expressão de genes associados ao fenótipo tronco e diminuiu a formação de tumoresferas. A inibição da expressão de IKKß em células A549 reduziu também a capacidade de autorrenovação de CITs. Estes resultados sugerem que IKKß desempenha um papel importante na manutenção do fenótipo tronco-tumoral de CITs pulmonares induzidas por KRAS. Em seguida, nós demonstramos que a inibição da atividade de IKKß afetou preferencialmente a proliferação celular e o crescimento clonogênico de células oriundas da cultura de tumoresfera, sugerindo que IKKß desempenha um papel mais importante em CITs do que em células derivadas da cultura aderente. A análise por citometria de fluxo identificou que células derivadas da cultura de tumoresfera apresentam um enriquecimento para células CD24+ na linhagem A549 e células CD44+ na linhagem H358, sugerindo que estes possam ser marcadores promissores para purificação de CITs nestas linhagens. Adicionalmente, demonstramos, por ensaios de wound-healing de células A549 e H358, que a inibição da atividade de IKKß reduziu a migração celular, uma outra uma propriedade aumentada em CITs. Além disso, mostramos que a atividade da quinase IKKß em células A549 e H358 não depende das vias da MAPK ou PI3K/Akt. Interessantemente, a inibição combinada de IKK (um efetor downstream de KRAS) e de EGFR/ERRB2 (reguladores upstream de KRAS que ativam as vias MAPK e PI3K/Akt) reduziu de forma aditiva a formação de tumoresferas, proliferação e migração celular. Quando avaliados em conjunto, nossos resultados sugerem que a quinase IKKß desempenha um papel importante na biologia de CITs pulmonares portadoras de KRAS oncogênica e que a inibição desta quinase sozinha ou em combinação com a inibição de outras vias pode representar uma estratégia terapêutica promissora a ser explorada para reduzir a recidiva e metástase no câncer de pulmão induzido por KRAS

The most frequent genetic alterations in lung cancer are point mutations that activate the KRAS oncogene. Although these mutations are causally related to oncogenesis, different approaches to inhibit RAS proteins directly have not been successful to date. Therefore, for better therapeutic targets for lung cancer to become available, it is necessary to identify the molecular mechanisms activated by KRAS that are directly involved with important malignant features, such as the development and maintenance of a cancer stem-like phenotype by the tumour-initiating cells (TICs). TICs, also known as cancer stem cells, are defined as a subpopulation of tumour cells able to self-renew, promote tumour initiation, and sustain tumour growth. The development of therapeutic strategies to target these cells is imperative to improve the efficacy of antitumor therapy. Since KRAS is associated with the maintenance of a cancer stem-like phenotype and activates the transcription factor NF-kB through the IKKß kinase to promote lung tumourigenesis, we hypothesised that IKKß kinase contributes to the cancer stem-like phenotype induced by KRAS in lung cancer. We used tumoursphere formation assays to enrich and evaluate the function of TICs of KRAS-mutant cell lines A549 and H358. A549 and H358 cells formed tumourspheres in low adhesion culture and, when compared to cells grown in adherent culture, sphere-derived cells displayed increased clonogenic growth, higher expression of stemness genes by qPCR, and increased IKKß kinase activity . Inhibition of IKKß activity through a highly specific pharmacological inhibitor (Compound A) slightly decreased proliferation of A549 and H358 cells without inducing significant cell death. On the other hand, inhibition of IKKß activity or expression by RNA interference reduced the expression of stemness genes and decreased tumoursphere formation. Inhibition of IKKß expression in A549 cells also reduced TICs self-renewal . These results suggest that IKKß plays an important role in maintaining the cancer stem-like phenotype of KRAS-driven lung TICs. Next, we demonstrated that IKKß inhibition preferentially reduced cell proliferation and clonogenic growth of sphere-derived cells, suggesting that IKKß plays a more important role in TICs than in adherent culture-derived cells. Flow cytometry analysis identified that sphere-derived cells display an enrichment for the surface marker CD24 in A549 cells and CD44 in H358 cells, indicating that these could be promising markers for the purification of TICs in these cell lines. Furthermore, we have shown by wound-healing assays of A549 and H358 cells that IKKß inhibition reduced cell migration , another feature increased in TICs. In addition, we have shown that IKKß activity in A549 and H358 cells does not depend on the MAPK or PI3K/Akt pathways. Interestingly, combined inhibition of IKKß (a downstream effector of KRAS) and EGFR/ERBB2 (upstream regulators of KRAS that activate the MAPK and PI3K/Akt pathways) additively reduced tumoursphere formation, cell proliferation and migration. Taken together, our results suggest that IKKß kinase plays an important role in the biology of KRAS-driven lung TICs, and that inhibition of this kinase alone or in combination with inhibition of other signalling pathways may represent a promising therapeutic strategy to be explored in order to reduce tumour recurrence and metastasis in KRAS-driven lung cancer

Spatial distribution of cancer stem cells in head and neck squamous cell carcinomas.

BACKGROUND: CD44 and aldehyde dehydrogenase 1 (ALDH1) are considered putative markers of highly tumorigenic cells (i.e., cancer stem-like cells) in head and neck squamous cell carcinomas. This small subset of cells is believed to be the primary responsible for tumor initiation and progression. The objectives of this study were (i) to evaluate the patterns of CD44 and ALDH1 expression in the tumor center and in the invasive front, as well as in adjacent non-tumor epithelium, and (ii) to correlate these findings with clinical parameters. MATERIALS AND METHODS: The sample comprised 44 patients with primary head and neck squamous cell carcinomas. Hematoxylin and eosin (HE) staining was used for histopathological tumor grading and for morphological analysis of adjacent non-tumor epithelium. Semiquantitative analysis was performed in histological sections immunostained for CD44 and ALDH1. RESULTS: ALDH1 immunostaining in the invasive front showed positive association with tumor size, regional metastasis, tumor histopathological grading, and disease progression. Moreover, expression of this marker in both tumor invasive front and adjacent non-tumor epithelium was related with more aggressive tumors. CD44 immunostaining was heterogeneous in all areas evaluated and did not show association with clinical data. CONCLUSION: Collectively, these data suggest that ALDH1 immunostaining in the invasive front and in adjacent non-tumor epithelium may help identify tumors with a more aggressive behavior, potentially contributing to improving treatment customization and the monitoring of patients with head and neck cancer.

Presença de marcadores de células-tronco em linhagens celulares humanas de câncer de mama: possível valor em diagnóstico, prognóstico e terapía / Analysis of stem cells markers in human breast cancer cell lines

O câncer de mama é a doença maligna que mais acomete as mulheres no mundo. Apesar dos inúmeros tratamentos, o óbito se deve principalmente à doença metastática que pode se desenvolver a partir do tumor primário. Esta progressão tumoral decorre da dificuldade de se estabelecer um prognóstico mais preciso. Atualmente, a teoria de células iniciadoras de tumor vem sendo estudada para tentar explicar a biologia do câncer e descrever novos alvos para prognósticos e terapias. O carcinoma mamário foi o primeiro tumor sólido para o qual foi identificada uma subpopulação celular, definida como CD44+/CD24-, apresentando as características de células iniciadoras tumorais. Embora este fenótipo venha sendo muito utilizado para descrever as células iniciadoras tumorais de mama, muitos trabalhos tem questionado a relevância clínica desses marcadores, enfatizando que outros marcadores devem ser identificados. Assim, o objetivo deste trabalho é analisar e caracterizar marcadores de células-tronco que possam estar relacionados com o grau de malignidade no modelo de câncer de mama. Inicialmente, analisou-se a expressão de 10 marcadores de células-tronco em diferentes linhagens de câncer de mama que apresentam graus crescentes de malignidade. O CD90 foi selecionado devido à alta expressão desse marcador na linhagem mais agressiva Hs578T. Para a caracterização deste marcador, realizou-se ensaios funcionais, através do silenciamento do CD90 na linhagem tumorigênica Hs579T e sua superexpressão na linhagem não-tumorigênica MCF10A. As linhagens celulares geradas foram caracterizadas quanto ao crescimento celular, potencial invasivo e metastático. Foi possível observar que houve uma alteração da morfologia nas linhagens transformadas com o CD90 e, também, um maior tempo de dobramento na linhagem Hs578T-CD90- e um menor na MCF10A-CD90+. Além disso, a linhagem MCF10-CD90+ foi capaz de crescer independentemente de EGF. Através da análise da via EGF, foi possível observar que houve um aumento da expressão da forma fosforilada do receptor e dos fatores Erk, c-Jun, e Jnk na linhagem MCF10A-CD90+ e uma diminuição dos mesmos na linhagem Hs578T-CD90-. A análise da atividade do elemento responsivo do fator de transcrição AP1 comprovou que a via de EGF é funcional na linhagem MCF10-CD90+. Também foram analisados os marcadores de transição epitélio-mesenquimal, verificando-se aumento da expressão dos marcadores mesenquimais na linhagem MCF10A-CD90+ e diminuição na linhagem Hs578T-CD90-. Os ensaios in vitro de invasão mostraram que as células MCF10-CD90+ são capazes de migrar e invadir e as células Hs578T-CD90- apresentam diminuição significativa da habilidade de migração e invasão. Além disso, os ensaios de metástase in vitro e in vivo, mostraram que a superexpressão de CD90 levou à malignização das células MCF10A. Por outro lado, a linhagem Hs578T-CD90- apresentou menor potencial metastático in vitro. Portanto, neste trabalho, pela primeira vez, o CD 90 foi caracterizado funcionalmente como um marcador envolvido na transformação maligna do carcinoma mamário, contribuindo, assim, para melhor entendimento da biologia do câncer de mama e para que se possa desenvolver novas ferramentas de diagnóstico/prognóstico e novos protocolos clínicos e terapêuticos

Breast cancer is the malignant disease which affects the highest number of women in the world. In spite of the numerous treatments available, death is primarily due to the metastatic disease that may develop from the primary tumor. This tumor progression occurs because of the difficulty in establishing an accurate diagnosis/prognosis. Currently, the tumor initiating cells theory is being applied in an attempt to explain cancer biology and to unveil new diagnostic and therapeutic targets. Mammary carcinoma was the first solid tumor in which a cellular subpopulation, defined as CD44+/CD24-, was associated with tumor initiating cells. Although this phenotype has been widely used to describe breast tumor initiating cells, several studies have questioned the clinical relevance of these markers, emphasizing that additional markers should be identified. The objective of the present study is to analyze and characterize stem cell markers that may be related to malignancy stages in the breast cancer model. Initially, the expression of 10 stem cell markers was analyzed in different breast cancer cell lines displaying different malignancy grades. CD90 was selected due to its high expression levels in the most aggressive cell line, namely: Hs578T. In order to further characterize this marker, a functional study was performed in which CD90 was silenced in the Hs578T tumorigenic cell line and overexpressed in the non-tumorigenic MCF10A cell line. The resulting cell lines were characterized relative to growth rate and invasive and metastatic potential. A change in morphology readily was observed in the cell lines overexpressing CD90. In addition, the Hs578T-CD90-cell line presented an increased doubling time (DT), while the MCF10A-CD90+ cell line displayed a lower DT.. Furthermore, MC10-CD90+ cells were able to grow in the absence of EGF. Analysis of components of the EGF pathwayrevealed increased expression levels of the phosphorylated form of Erk, c-Jun and Jnk receptors in the MCF10-CD90+ cell line, while Hs578T-CD90- cells presented decreased expression of the same factors and receptors. Analysis of the activity of the AP1 responsive element allowed confirmation that the EGF pathway is functional in the MCF10-CD90+. . Epithelial-mesenquimal transition markers presented increased expression levels in the MCF10A-CD90+ cell line, accompanied by decreased expression levels in Hs578T-CD90- cells. In vitro invasion assays showed that MCF10A-CD90+ cells are capable of migrating and invading, while Hs578T-CD90- cells presented a significant decrease in their ability to migrate and invade. Additionally, in vitro and in vivo metastasis assays showed that malignization ensued upon overexpression of CD90 in MCF10A cells and a lower tendency to form metastasis in vitro was observed for the Hs578T-CD90- cell line. Therefore, the present study presents, for the first time in the literature, the functional characterization of CD90 as a genetic marker involved in the malignant transformation of mammary carcinoma, leading to a better understanding of the breast cancer biology, which may, in turn, lead to the development of new clinical and therapeutic protocols

Cancer stem cells - the current status of an old concept: literature review and clinical approaches

As regards their morphology and biology, tumours consist of heterogeneous cell populations. The cancer stem cell (CSC) hypothesis assumes that a tumour is hierarchically organized and not all of the cells are equally capable of generating descendants, similarly to normal tissue. The only cells being able to self-renew and produce a heterogeneous tumour cell population are cancer stem cells. CSCs probably derive from normal stem cells, although progenitor cells may be taken into consideration as the source of cancer stem cells. CSCs reside in the niche defined as the microenvironment formed by stromal cells, vasculature and extracellular matrix. The CSC assays include FACS sorting, xenotransplantation to immunodeficient mice (SCID), incubation with Hoechst 33342 dye, cell culture in non-adherent conditions, cell culture with bromodeoxyuridine. CSCs have certain properties that make them resistant to anticancer therapy, which suggests they may be the target for potential therapeutic strategies.