Modulation of tumor plasticity by senescent cells: Deciphering basic mechanisms and survival pathways to unravel therapeutic options.

Senescence is a cellular state in which the cell loses its proliferative capacity, often irreversibly. Physiologically, it occurs due to a limited capacity of cell division associated with telomere shortening, the so-called replicative senescence. It can also be induced early due to DNA damage, oncogenic activation, oxidative stress, or damage to other cellular components (collectively named induced senescence). Tumor cells acquire the ability to bypass replicative senescence, thus ensuring the replicative immortality, a hallmark of cancer. Many anti-cancer therapies, however, can lead tumor cells to induced senescence. Initially, this response leads to a slowdown in tumor growth. However, the longstanding accumulation of senescent cells (SnCs) in tumors can promote neoplastic progression due to the enrichment of numerous molecules and extracellular vesicles that constitutes the senescence-associated secretory phenotype (SASP). Among other effects, SASP can potentiate or unlock the tumor plasticity and phenotypic transitions, another hallmark of cancer. This review discusses how SnCs can fuel mechanisms that underlie cancer plasticity, like cell differentiation, stemness, reprogramming, and epithelial-mesenchymal transition. We also discuss the main molecular mechanisms that make SnCs resistant to cell death, and potential strategies to target SnCs. At the end, we raise open questions and clinically relevant perspectives in the field.

Subcellular structure, heterogeneity, and plasticity of senescent cells.

Cellular senescence is a state of permanent growth arrest. It can be triggered by telomere shortening (replicative senescence) or prematurely induced by stresses such as DNA damage, oncogene overactivation, loss of tumor suppressor genes, oxidative stress, tissue factors, and others. Advances in techniques and experimental designs have provided new evidence about the biology of senescent cells (SnCs) and their importance in human health and disease. This review aims to describe the main aspects of SnCs phenotype focusing on alterations in subcellular compartments like plasma membrane, cytoskeleton, organelles, and nuclei. We also discuss the heterogeneity, dynamics, and plasticity of SnCs' phenotype, including the SASP, and pro-survival mechanisms. We advance on the multiple layers of phenotypic heterogeneity of SnCs, such as the heterogeneity between inducers, tissues and within a population of SnCs, discussing the relevance of these aspects to human health and disease. We also raise the main challenges as well alternatives to overcome them. Ultimately, we present open questions and perspectives in understanding the phenotype of SnCs from the perspective of basic and applied questions.

The rational modulation of autophagy sensitizes colorectal cancer cells to 5-fluouracil and oxaliplatin.

Colorectal cancer (CRC) is the third most common and deadliest cancer globally. Regimens using 5-fluorouracil (5FU) and Oxaliplatin (OXA) are the first-line treatment for CRC, but tumor recurrence is frequent. It is plausible to hypothesize that differential cellular responses are triggered after treatments depending on the genetic background of CRC cells and that the rational modulation of cell tolerance mechanisms like autophagy may reduce the regrowth of CRC cells. This study proposes investigating the cellular mechanisms triggered by CRC cells exposed to 5FU and OXA using a preclinical experimental design mimicking one cycle of the clinical regimen (i.e., 48 h of treatment repeated every 2 weeks). To test this, we treated CRC human cell lines HCT116 and HT29 with the 5FU and OXA, combined or not, for 48 h, followed by analysis for two additional weeks. Compared to single-drug treatments, the co-treatment reduced tumor cell regrowth, clonogenicity and stemness, phenotypes associated with tumor aggressiveness and poor prognosis in clinics. This effect was exerted by the induction of apoptosis and senescence only in the co-treatment. However, a week after treatment, cells that tolerated the treatment had high levels of autophagy features and restored the proliferative phenotype, resembling tumor recurrence. The pharmacologic suppression of early autophagy during its peak of occurrence, but not concomitant with chemotherapeutics, strongly reduced cell regrowth. Overall, our experimental model provides new insights into the cellular mechanisms that underlie the response and tolerance of CRC cells to 5FU and OXA, suggesting optimized, time-specific autophagy inhibition as a new avenue for improving the efficacy of current treatments.

Ex vivo gene therapy for lysosomal storage disorders: future perspectives.

INTRODUCTION: Lysosomal storage disorders (LSD) are a group of monogenic rare diseases caused by pathogenic variants in genes that encode proteins related to lysosomal function. These disorders are good candidates for gene therapy for different reasons: they are monogenic, most of lysosomal proteins are enzymes that can be secreted and cross-correct neighboring cells, and small quantities of these proteins are able to produce clinical benefits in many cases. Ex vivo gene therapy allows for autologous transplant of modified cells from different sources, including stem cells and hematopoietic precursors. AREAS COVERED: Here, we summarize the main gene therapy and genome editing strategies that are currently being used as ex vivo gene therapy approaches for lysosomal disorders, highlighting important characteristics, such as vectors used, strategies, types of cells that are modified and main results in different disorders. EXPERT OPINION: Clinical trials are already ongoing, and soon approved therapies for LSD based on ex vivo gene therapy approaches should reach the market.

Molecular markers associated with the outcome of tamoxifen treatment in estrogen receptor-positive breast cancer patients: scoping review and in silico analysis.

Tamoxifen (TMX) is used as adjuvant therapy for estrogen receptor-positive (ER+) breast cancer cases due to its affinity and inhibitory effects. However, about 30% of cases show drug resistance, resulting in recurrence and metastasis, the leading causes of death. A literature review can help to elucidate the main cellular processes involved in TMX resistance. A scoping review was performed to find clinical studies investigating the association of expression of molecular markers profiles with long-term outcomes in ER+ patients treated with TMX. In silico analysis was performed to assess the interrelationship among the selected markers, evaluating the joint involvement with the biological processes. Forty-five studies were selected according to the inclusion and exclusion criteria. After clustering and gene ontology analysis, 23 molecular markers were significantly associated, forming three clusters of strong correlation with cell cycle regulation, signal transduction of proliferative stimuli, and hormone response involved in morphogenesis and differentiation of mammary gland. Also, it was found that overexpression of markers in selected clusters is a significant indicator of poor overall survival. The proposed review offered a better understanding of independent data from the literature, revealing an integrative network of markers involved in cellular processes that could modulate the response of TMX. Analysis of these mechanisms and their molecular components could improve the effectiveness of TMX.

Simplified low-cost methodology to establish, histologically process and analyze three-dimensional cancer cell spheroid arrays.

Differently of two-dimensional cell culture, three-dimensional (3D) multicellular spheroid model allows cells to establish cell-cell/cell-matrix interactions over the entire cell surface, more closely mimicking tumor microenvironments and cellular subpopulations with specific standards of morphology, differentiation and gene expression. Thenceforth several methodologies involving or the 3D cell aggregates generation or its histological processing and analysis have emerged, but in general they are laborious, expensive and complex to set up as a routine technique. Thus, we developed a complete methodology, detailing a simple, accessible and low-cost step by step, including 1) the 3D cell aggregate generation using hanging drop technique; 2) providing a simple way to assess morphological parameters of generated spheroids; followed by 3) a multiple and organized histological processing, keeping several individual spheroids inside an agarose apparatus, maintaining a known order and position of each ones, similar to tissue microarray principle; 4) until the last step, where it is allowed a simultaneous histological composition analysis of several spheroid slices, organized side by side, in a same block section, through conventional stainings or 5) immunostaining against different molecular markers. Therefore, the present methodology aims to popularize 3D cell culture, allowing to make this a regular technique in basic cell biology research, once all steps are performed without using onerous reagents, materials or equipment. In addition to bring the agarose apparatus as a simple low cost novelty, allowing high-throughput analysis of several spheroids simultaneously in an organized manner.

Maternal feeding associated to post-weaning diet affects metabolic and behavioral parameters in female offspring.

Genetic and environmental factors related to maternal diet may predispose offspring to serious diseases. However, consequences of a maternal diet intervention during gestation and lactation, and its association with caloric restriction after weaning on the progeny are not completely known. In this context, the goal of the present study was to investigate how different maternal diets, control (CONT), hypercaloric (HD) or restrictive (RD) diets during gestation and lactation, may affect the metabolism and behavior of the offspring that was also submitted to RD. Experimental groups were abbreviated accordingly maternal/offspring diets: CONT/CONT, CONT/RD, RD/CONT, RD/RD, HD/CONT, HD/RD. Our results showed that glucose serum concentration is increased in mice from dams fed a HD. However, offspring from RD-fed dams showed lower insulin and leptin levels than the other groups, indicating a maternal diet effect. Moreover, animals from RD/CONT group showed a higher adipocyte area in comparison to both HD/CONT and CON/CONT. Offspring from RD-fed dams exhibited a decrease in lateral area locomotion in the open field test. Evaluation of anxiety-like behavior and recognition memory showed no significant difference among groups. Thus, maternal RD provides a beneficial response in metabolic parameters, but its effects on behavior is not completely clarified.

Low Dose of Doxorubicin Potentiates the Effect of Temozolomide in Glioblastoma Cells.

Glioblastoma (GBM) is an aggressive brain tumor with temozolomide (TMZ)-based chemotherapy as the main therapeutic strategy. Doxorubicin (DOX) is not used in gliomas due to its low bioavailability in the brain; however, new delivery strategies and low doses may be effective in the long term, especially as part of a drug cocktail. Our aim was to evaluate the chronic effects of low doses of DOX and TMZ in GBM. Human U87-ATCC cells and a primary GBM culture were chronically treated with TMZ (5 µM) and DOX (1 and 10 nM) alone or combined. DOX resulted in a reduction in the number of cells over a period of 35 days and delayed the cell regrowth. In addition, DOX induced cell senescence and reduced tumor sphere formation and the proportion of NANOG- and OCT4-positive cells after 7 days. Low doses of TMZ potentiated the effects of DOX on senescence and sphere formation. This combined response using low doses of DOX may pave the way for its use in glioma therapy, with new technologies to overcome its low blood-brain barrier permeability.

Vinblastine and antihelmintic mebendazole potentiate temozolomide in resistant gliomas.

Glioblastoma (GBM) is a very aggressive tumor that has not had substantial therapeutic improvement since the introduction of temozolomide (TMZ) in combination with radiotherapy. Combining TMZ with other chemotherapeutic agents is a strategy that could be further explored for GBM. To search for molecular predictors of TMZ resistance, the TCGA (The Cancer Genome Atlas) database was utilized to assess the impact of specific genes on TMZ response. Patients whose tumors expressed low levels of FGFR3 and AKT2 responded poorly to TMZ. Combination treatment of vinblastine (VBL) plus mebendazole (MBZ) with TMZ was more effective in reducing cell number in most cultures when compared to TMZ alone, especially in cells with low expression levels of FGFR3 and AKT2. Cell cycle distribution and nuclear morphometric analysis indicated that the triple combination of TMZ, VBL and MBZ (TVM) was able to induce polyploidy and senescence, in addition to increasing the Notch3 RNA level in patient-derived gliomas. Thus, this set of data suggests that the triple combination of TMZ, VBL and MBZ may be a considerable therapeutic alternative for the TMZ-tolerant gliomas that harbor low expression of FGFR3/AKT2.

The regrowth kinetic of the surviving population is independent of acute and chronic responses to temozolomide in glioblastoma cell lines.

Chemotherapy acts on cancer cells by producing multiple effects on a cell population including cell cycle arrest, necrosis, apoptosis and senescence. However, often a subpopulation of cells survives and the behavior of this subpopulation, which is responsible for cancer recurrence, remains obscure. Here we investigated the in vitro short- and long-term responses of six glioblastoma cell lines to clinically relevant doses of temozolomide for 5 days followed by 23 days of recovery, mimicking the standard schedule used in glioblastoma patient for this drug. These cells presented different profiles of sensitivity to temozolomide with varying levels of cell cycle arrest, autophagy and senescence, followed by a regrowth of the surviving cells. The initial reduction in cell number and the subsequent regrowth was analyzed with four new parameters applied to Cumulative Population Doubling (CPD) curves that describe the overall sensitivity of the population and the characteristic of the regrowth: the relative end point CPD (RendCPD); the relative Area Under Curve (rAUC); the Relative Time to Cross a Threshold (RTCT); and the Relative Proliferation Rate (RPR). Surprisingly, the kinetics of regrowth were not predicted by the mechanisms activated after treatment nor by the acute or overall sensitivity. With this study we added new parameters that describe key responses of glioblastoma cell populations to temozolomide treatment. These parameters can also be applied to other cell types and treatments and will help to understand the behavior of the surviving cancer cells after treatment and shed light on studies of cancer resistance and recurrence.

Analysis of the safety of mesenchymal stromal cells secretome for glioblastoma treatment.

BACKGROUND AIMS: The purpose of this study was to investigate whether the secretome of human adipose-derived stem cells (hADSC) affects human glioblastoma (GBM) cancer stem cell (CSC) subpopulation or has any influence on drug resistance and cell migration, evaluating the safety of hADSCs for novel cancer therapies. METHODS: hADSCs were maintained in contact with fresh culture medium to produce hADSCs conditioned medium (CM). GBM U87 cells were cultured with CM and sphere formation, expression of genes related to resistance and CSCs-MGMT, OCT4, SOX2, NOTCH1, MSI1-and protein expression of OCT4 and Nanog were analyzed. The influence of hADSC CM on GBM resistance to temozolomide (TMZ) was evaluated by measuring cumulative population doubling and hADSC CM influence on tumor cell migration was analyzed using transwell assay. RESULTS: hADSC CM did not alter CSC-related features such as sphere-forming capacity and expression of genes related to CSC. hADSC CM treatment alone did not change proliferation rate of U87 cells and, most important, did not alter the response of tumor cells to TMZ. However, hADSC CM secretome increased the migration capacity of glioblastoma cells. DISCUSSION: hADSC CM neither induced an enrichment of CSCs in U87 cells population nor interfered in the response to TMZ in culture. Nevertheless, paracrine factors released by hADSCs were able to modulate glioblastoma cells migration. These findings provide novel information regarding the safety of using hADSCs against cancer and highlight the importance of considering hADSC-tumor cells interactions in tumor microenvironment in the design of novel cell therapies.