Natural killer cell-based adoptive immunotherapy eradicates and drives differentiation of chemoresistant bladder cancer stem-like cells.

BACKGROUND: High-grade non-muscle invasive bladder cancer (NMIBC) has a high risk of recurrence and progression to muscle-invasive forms, which seems to be largely related to the presence of tumorigenic stem-like cell populations that are refractory to conventional therapies. Here, we evaluated the therapeutic potential of Natural Killer (NK) cell-based adoptive immunotherapy against chemoresistant bladder cancer stem-like cells (CSCs) in a pre-clinical relevant model, using NK cells from healthy donors and NMIBC patients. METHODS: Cytokine-activated NK cells from healthy donors and from high-grade NMIBC patients were phenotypically characterized and assayed in vitro against stem-like and bulk differentiated bladder cancer cells. Stem-like cells were isolated from two bladder cancer cell lines using the sphere-forming assay. The in vivo therapeutic efficacy was evaluated in mice bearing a CSC-induced orthotopic bladder cancer. Animals were treated by intravesical instillation of interleukin-activated NK cells. Tumor response was evaluated longitudinally by non-invasive bioluminescence imaging. RESULTS: NK cells from healthy donors upon activation with IL-2 and IL-15 kills indiscriminately both stem-like and differentiated tumor cells via stress ligand recognition. In addition to cell killing, NK cells shifted CSCs towards a more differentiated phenotype, rendering them more susceptible to cisplatin, highlighting the benefits of a possible combined therapy. On the contrary, NK cells from NMIBC patients displayed a low density on NK cytotoxicity receptors, adhesion molecules and a more immature phenotype, losing their ability to kill and drive differentiation of CSCs. The local administration, via the transurethral route, of activated NK cells from healthy donors provides an efficient tumor infiltration and a subsequent robust tumoricidal activity against bladder cancer with high selective cytolytic activity against CSCs, leading to a dramatic reduction in tumor burden from 80 % to complete remission. CONCLUSION: Although pre-clinical, our results strongly suggest that an immunotherapeutic strategy using allogeneic activated NK cells from healthy donors is effective and should be exploited as a complementary therapeutic strategy in high-risk NMIBC patients to prevent tumor recurrence and progression.

The need for transparency and good practices in the qPCR literature.

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

Effect of chronic methylphenidate treatment on hippocampal neurovascular unit and memory performance in late adolescent rats.

Methylphenidate (MPH) is the classic treatment for attention deficit hyperactivity disorder (ADHD) among children and adults. Despite its beneficial effects, non-medical use of MPH is nowadays a problem with high impact on society. Thus, our goal was to uncover the neurovascular and cognitive effects of MPH chronic use during a critical period of development in control conditions. For that, male Wistar Kyoto rats were treated with MPH (1.5 or 5 mg/kg/day at weekdays, per os) from P28 to P55. We concluded that the higher dose of MPH caused hippocampal blood-brain barrier (BBB) hyperpermeability by vesicular transport (transcytosis) concomitantly with the presence of peripheral immune cells in the brain parenchyma. These observations were confirmed by in vitro studies, in which the knockdown of caveolin-1 in human brain endothelial cells prevented the increased permeability and leukocytes transmigration triggered by MPH (100 µM, 24 h). Furthermore, MPH led to astrocytic atrophy and to a decrease in the levels of several synaptic proteins and impairment of AKT/CREB signaling, together with working memory deficit assessed in the Y-maze test. On the contrary, we verified that the lower dose of MPH (1.5 mg/kg/day) increased astrocytic processes and upregulated several neuronal proteins as well as signaling pathways involved in synaptic plasticity culminating in working memory improvement. In conclusion, the present study reveals that a lower dose of MPH in normal rats improves memory performance being associated with the modulation of astrocytic morphology and synaptic machinery. However, a higher dose of MPH leads to BBB dysfunction and memory impairment.

Leukemic Stem Cells S(p)liced Off.

Myeloid malignancies often arise from an aging hematopoietic system and are currently incurable due to therapy-resistant, disease-reinitiating leukemic stem cells. In this issue of Cell Stem Cell, Crews et al. (2016) report RNA splice isoform signatures unique to patient-derived leukemic stem cells that constitute a therapeutic Achilles' heel of myeloid leukemia.

Functional and molecular characterization of cancer stem-like cells in bladder cancer: a potential signature for muscle-invasive tumors.

Striking evidence associates cancer stem cells (CSCs) to the high recurrence rates and poor survival of patients with muscle-invasive bladder cancer (BC). However, the prognostic implication of those cells in risk stratification is not firmly established, mainly due to the functional and phenotypic heterogeneity of CSCs populations, as well as, to the conflicting data regarding their identification based on a single specific marker. This emphasizes the need to exploit putative CSC-related molecular markers with potential prognostic significance in BC patients.This study aimed to isolate and characterize bladder CSCs making use of different functional and molecular approaches. The data obtained provide strong evidence that muscle-invasive BC is enriched with a heterogeneous stem-like population characterized by enhanced chemoresistance and tumor initiating properties, able to recapitulate the heterogeneity of the original tumor. Additionally, a logistic regression analysis identified a 2-gene stem-like signature (SOX2 and ALDH2) that allows a 93% accurate discrimination between non-muscle-invasive and invasive tumors.Our findings suggest that a stemness-related gene signature, combined with a cluster of markers to more narrowly refine the CSC phenotype, could better identify BC patients that would benefit from a more aggressive therapeutic intervention targeting CSCs population.

Drug transporters play a key role in the complex process of Imatinib resistance in vitro.

Imatinib resistance has been associated with BCR-ABL alterations, but other mechanisms might be involved, like drug transporters. Additionally, the impact of poor adherence in resistance has been little explored. Using sensitive and resistance CML cell lines, we investigated the expression of influx/efflux transporters, like P-gP and OCT1. In the therapeutic interruption model, we observed decrease of influx and increase in efflux transporters combined with BCR-ABL over-expression. Comparatively, resistant cells obtained by continuous TKI exposure only demonstrated alterations in drug's transporters. By exploring P-gP expression of resistant cells, we observed the potential of P-gP inhibitor in circumventing Imatinib resistance. Our results revealed the importance of treatment interruptions for expected response levels and show the complexity of Imatinib resistant process. Efflux transports appear as not only relevant for acquisition of resistant phenotype, but also as valid therapeutic tool for managing resistance.