Phenotypic, structural, and ultrastructural analysis of triple-negative breast cancer cell lines and breast cancer stem cell subpopulation.

Triple negative breast cancer (TNBC) is a highly heterogeneous disease, which influences the therapeutic response and makes difficult the discovery of effective targets. This heterogeneity is attributed to the presence of breast cancer stem cells (BCSCs), which determines resistance to chemotherapy and subsequently disease recurrence and metastasis. In this context, this work aimed to evaluate the morphological and phenotypic cellular heterogeneity of two TNBC cell lines cultured in monolayer and tumorsphere (TS) models by fluorescence and electron microscopy and flow cytometry. The BT-549 and Hs 578T analyses demonstrated large phenotypic and morphological heterogeneity between these cell lines, as well as between the cell subpopulations that compose them. BT-549 and Hs 578T are heterogeneous considering the cell surface marker CD44 and CD24 expression, characterizing BCSC mesenchymal-like cells (CD44+/CD24-), epithelial cells (CD44-/CD24+), hybrid cells with mesenchymal and epithelial features (CD44+/CD24+), and CD44-/CD24- cells. BCSC epithelial-like cells (ALDH+) were found in BT-549, BT-549 TS, and Hs 578T TS; however, only BT-549 TS showed a high ALDH activity. Ultrastructural characterization showed the heterogeneity within and among BT-549 and Hs 578T in monolayer and TS models being formed by more than one cellular type. Further, the mesenchymal characteristic of these cells is demonstrated by E-cadherin absence and filopodia. It is well known that tumor cell heterogeneity can influence survival, therapy responses, and the rate of tumor growth. Thus, molecular understanding of this heterogeneity is essential for the identification of potential therapeutic options and vulnerabilities of oncological patients.

STAT3 as a promising chemoresistance biomarker associated with the CD44+/high/CD24-/low/ALDH+ BCSCs-like subset of the triple-negative breast cancer (TNBC) cell line.

The cancer stem cell (CSC) concept is currently employed to explain the mechanism of multidrug resistance that is implicated in the reduced efficacy of many chemotherapeutic agents, consequently leading to metastatic spread and disease relapse. We searched for potential predictive markers of doxorubicin (DOX) resistance in breast cancer stem cells (BCSCs) of the BT-549 human triple-negative breast cancer (TNBC) cell line classified as a claudin-low subtype. In this study, we show that BT-549 presents a BCSCs-like subset determined by a CD44+/high/CD24-/low/ALDH1+ phenotype. The CD44+/high/CD24-/low/ALDH+ BCSCs-like subset presented the downregulation of a majority of the genes analyzed (64 genes), and only 3 genes were upregulated after DOX treatment. Among the upregulated genes, MAPK3, PRKCZ and STAT3, STAT3 presented a higher level of upregulation in the DOX-treated CD44+/high/CD24-/low/ALDH+ BCSCs-like subset. The identification of biomarkers that predict antitumor responses is at the top of cancer research priorities. STAT3 was highlighted as a molecular signature in the CD44+/high/CD24-/low/ALDH1+ BCSCs-like subset obtained from the TNBC BT-549 cell line related to DOX resistance. A majority of the evaluated genes in the EGF pathway appear to be not associated with DOX resistance, as observed in the CD44+/high/CD24-/low/ALDH1+ BCSCs-like subset.

In vitro three-dimensional microenvironment induces differential gene expression ofbreast cancer biomarkers / Microambiente tridimensional in vitro induz a expressâo gênica diferencial de biomarcadores do câncer de mama

In vitro three-dimensional culture has an invaluable role in the study of cell biology because it can provide a more physiologic microenvironment than the conventional two-dimensional culture, much more similar to in vivo features. The present study aimed to assess the expression ofbreast cancer biomarkers (ERBB2, KRT5,MKI67, CDH3 and TP63) in breast cancer cell lines (BT-549 and Hs 578T) in these different in vitro culturing (2D x 3D) by quantitative PCR. The results showed a differential expression of those genes when the 2D cell lines were cultured in Matrigel. This analysis provides indications of their expression in Matrigel, highlighting ERBB2 and TP63 that emerge like promising markers of cancer stem cell. These findings ofter a better understanding ofthe biology of the breast cancer cell lines analyzed for further use of these models in drug cancer cytotoxicity assays.

O cultivo tridimensional in vitro tem um papel inestimável no estudo da biologia das células, pois proporciona um microambiente mais fisiológico do que a cultura bidimensional, muito mais semelhante a características in vivo. O presente estudo teve como objetivo avaliar a expressão de biomarcadores do câncer de mama (ERBB2, KRT5, MKI67, CDH3 and TP63) em linhagens celulares de câncer de mama (BT-549 e Hs 578T) em sistemas de cultura in vitro 2D x 3D por PCR quantitativo. Os resultados mostraram uma expressâo diferencial dos genes quando as linhagens celulares cultivadas em 2D foram cultivadas em Matrigel. Esta análise fornece indícios da expressão desses genes em Matrigel com destaque para ERBB2 e TP63, que surgem como promissores marcadores de células-tronco do câncer. Estes resultados oferecem uma melhor compreensão da biologia das linhagenscelulares de câncer de mama estudadas para futuramente utilizar esse modelo em ensaios de citotoxicidade contra o câncer.

The role of GADD45A in resistance to oxidative stress-mediated cell death in human colon tumor cell lines

Objective: GADD45A is a growth arrest-associated gene, directly involved in the maintenance of genomic stability. In fact, in the absence of this protein cells became sensitized to death by ultraviolet irradiation or cisplatin. However, this role with respect to cell damage triggered by Reactive Oxygen Species (ROS) is not well understood. Thus, in an attempt to evaluate its role in oxidative stress-induced apoptosis, we analyzed its expression after induction of ROS in human colon cell lines. Material and methods: Cell lines derived from human colon tumors (RKO-AS45-1 and HuTu-80) were treated with Menadione (MEN) and Hydrogen Peroxide (HP). Next, the expression of GADD45A was evaluated by semi-quantitative RT-PCR analysis. Results: The results indicated that expression of this gene was associated with resistance to apoptosis. Additionally, cells with high expression of GADD45A were resistant to treatment with oxidative stress-inducing compounds. Conclusion: In conclusion, we propose that the expression of the GADD45A gene can be used as a functional tool to predict cellular responses to antitumor treatments.

Sporulation and diversity of arbuscular mycorrhizal fungi in Brazil Pine in the field and in the greenhouse.

The aim of this work was to assess the sporulation and diversity of arbuscular mycorrhizal fungi (AMF) at different forest sites with Araucaria angustifolia (Bert.) O. Ktze. (Brazil Pine). In addition, a greenhouse experiment was carried out to test the use of traditional trap plants (maize + peanut) or A. angustifolia to estimate the diversity of AMF at each site. Soil samples were taken in two State Parks at southwestern Brazil: Campos do Jordão (Parque Estadual de Campos do Jordão [PECJ]) and Apiaí (Parque Estadual Turístico do Alto Ribeira [PETAR]), São Paulo State, in sites of either native or replanted forest. In PECJ, an extra site of replanted forest that was impacted by accidental fire and is now in a state of recuperation was also sampled. The spore densities and their morphological identification were compiled at each site. In the greenhouse, soil samples from each site were used as inoculum to promote spore multiplication on maize + peanut or A. angustifolia grown on a sandy, low-fertility substrate. Plants were harvested, respectively, after 4 months or 1 year of growth and assessed for mycorrhizal root colonization. Spore counts and identification were also performed in the substrate, after the harvest of plants. Twenty-five taxa were identified considering all sites. Species richness and diversity were greater in native forest areas, being Acaulospora, the genus with the most species. Differences in number of spores, diversity, and richness were found at the different sites of each State Park. Differences were also found when maize + peanut or A. angustifolia were used as trap plants. The traditional methodology using trap plants seems to underestimate the diversity of the AMF. The use of A. angustifolia as trap plant showed similar species richness to the field in PECJ, but the identified species were not necessarily the same. Nevertheless, for PETAR, both A. angustifolia and maize + peanut underestimated the species richness. Because the AMF sporulation can be affected by many conditions, it is impossible to draw detailed conclusions from this kind of survey. More precise experiments have to be set up to isolate the different factors that modulate the ecophysiological interactions between host plant and endophyte.