Laminin-332 sustains chemoresistance and quiescence as part of the human hepatic cancer stem cell niche.

BACKGROUND & AIMS: Cancer stem cells (CSCs) are thought to be persistent in tumours due to their chemoresistance and to cause relapse and metastasis. Hepatic carcinomas displaying hepatic progenitor cell (HPC) features have been associated with a poor prognosis, though it remains unclear how CSCs relate to these different histological subtypes. METHODS: Candidate CSCs were isolated using the side population (SP) technique from primary tissue samples diagnosed as keratin(K)19-negative or -positive hepatocellular carcinoma (HCC) or as combined hepatocellular/cholangiocarcinoma and analysed for gene and protein expression. The effect of laminin-332 was analysed in vitro by using HCC cell lines and in vivo using a xenograft mouse model. RESULTS: The size of the SP correlated with the degree of HPC features found in human hepatic cancer, and also showed an elevated mRNA expression of biliary/HPC markers and the extracellular matrix marker LAMC2, the gene encoding the laminin γ2-chain. Immunopositivity for the γ2-chain of laminin-332 was seen in the extracellular matrix surrounding small HPC-like tumour cells with a low proliferation rate. In vitro, laminin-332 increased K19 expression, phosphorylated mTOR and decreased phospho-histone H3 expression, indicating reduced cell mitosis. The effect of laminin-332 was enhanced upon mTORC1 inhibition and diminished when inhibiting mTORC1+C2. Resistance to doxorubicin and sorafenib treatment, and the SP fraction increased in the coated condition. In vivo, laminin-332 reduced tumour growth and sustained K19 expression. CONCLUSIONS: In this study we identified a prominent role for laminin-332 as part of the specialised CSC niche in maintaining and supporting cell 'stemness', which leads to chemoresistance and quiescence.

The neuroprotective effect of post ischemic brief mild hypothermic treatment correlates with apoptosis, but not with gliosis in endothelin-1 treated rats.

BACKGROUND: Stroke remains one of the most common diseases with a serious impact on quality of life but few effective treatments exist. Mild hypothermia (33°C) is a promising neuroprotective therapy in stroke management. This study investigated whether a delayed short mild hypothermic treatment is still beneficial as neuroprotective strategy in the endothelin-1 (Et-1) rat model for a transient focal cerebral ischemia. Two hours of mild hypothermia (33°C) was induced 20, 60 or 120 minutes after Et-1 infusion. During the experiment the cerebral blood flow (CBF) was measured via Laser Doppler Flowmetry in the striatum, which represents the core of the infarct. Functional outcome and infarct volume were assessed 24 hours after the insult. In this sub-acute phase following stroke induction, the effects of the hypothermic treatment on apoptosis, phagocytosis and astrogliosis were assessed as well. Apoptosis was determined using caspase-3 immunohistochemistry, phagocytic cells were visualized by CD-68 expression and astrogliosis was studied by glial fibrillary acidic protein (GFAP) staining. RESULTS: Cooling could be postponed up to 1 hour after the onset of the insult without losing its positive effects on neurological deficit and infarct volume. These results correlated with the caspase-3 staining. In contrast, the increased CD-68 expression post-stroke was reduced in the core of the insult with all treatment protocols. Hypothermia also reduced the increased levels of GFAP staining, even when it was delayed up to 2 hours after the insult. The study confirmed that the induction of the hypothermia treatment in the Et-1 model does not affect the CBF. CONCLUSIONS: These data indicate that in the Et-1 rat model, a short mild hypothermic treatment delayed for 1 hour is still neuroprotective and correlates with apoptosis. At the same time, hypothermia also establishes a lasting inhibitory effect on the activation of astrogliosis.

Mild hypothermia causes differential, time-dependent changes in cytokine expression and gliosis following endothelin-1-induced transient focal cerebral ischemia.

BACKGROUND: Stroke is an important cause of morbidity and mortality and few therapies exist thus far. Mild hypothermia (33°C) is a promising neuroprotective strategy to improve outcome after ischemic stroke. However, its complete mechanism of action has not yet been fully elaborated. This study is the first to investigate whether this neuroprotection occurs through modulation of the neuroinflammatory response after stroke in a time-dependent manner. METHODS: The Endothelin-1 (Et-1) model was used to elicit a transient focal cerebral ischemia in male Wistar rats. In this model, the core and penumbra of the insult are represented by the striatum and the cortex respectively. We assessed the effects of 2 hours of hypothermia, started 20 minutes after Et-1 injection on neurological outcome and infarct volume. Furthermore, pro- and anti-inflammatory cytokine expression was determined using ELISA. Microgliosis and astrogliosis were investigated using CD-68 and GFAP staining respectively. All parameters were determined 8, 24, 72 hours and 1 week after the administration of Et-1. RESULTS: Et-1 infusion caused neurological deficit and a reproducible infarct size which increased up to 3 days after the insult. Both parameters were significantly reduced by hypothermia. The strongest reduction in infarct volume with hypothermia, at 3 days, corresponded with increased microglial activation. Reducing the brain temperature affected the stroke induced increase in interleukin-1ß and tumor necrosis factor α in the striatum, 8 hours after its induction, but not at later time points. Transforming growth factor ß increased as a function of time after the Et-1-induced insult and was not influenced by cooling. Hypothermia reduced astrogliosis at 1 and 3 days after stroke onset. CONCLUSIONS: The beneficial effects of hypothermia after stroke on infarct volume and functional outcome coincide with a time-dependent modulation of the cytokine expression and gliosis.

The dual role of the neuroinflammatory response after ischemic stroke: modulatory effects of hypothermia.

Neuroinflammation is a key element in the ischemic cascade after cerebral ischemia that results in cell damage and death in the subacute phase. However, anti-inflammatory drugs do not improve outcome in clinical settings suggesting that the neuroinflammatory response after an ischemic stroke is not entirely detrimental. This review describes the different key players in neuroinflammation and their possible detrimental and protective effects in stroke. Because of its inhibitory influence on several pathways of the ischemic cascade, hypothermia has been introduced as a promising neuroprotective strategy. This review also discusses the influence of hypothermia on the neuroinflammatory response. We conclude that hypothermia exerts both stimulating and inhibiting effects on different aspects of neuroinflammation and hypothesize that these effects are key to neuroprotection.

Rapid clinical mutational testing of KRAS, BRAF and EGFR: a prospective comparative analysis of the Idylla technique with high-throughput next-generation sequencing.

AIMS: Precision medicine therapy is remodelling the diagnostic landscape of cancer. The success of these new therapies is often based on the presence or absence of a specific mutation in a tumour. The Idylla platform is designed to determine the mutational status of a tumour as quickly and accurately as possible, as a rapid, accurate diagnosis is of the utmost importance for the treatment of patients. This is the first complete prospective study to investigate the robustness of the Idylla platform for EGFR, KRAS and BRAF mutations in non-small cell lung cancer, metastatic colorectal cancer and metastatic melanoma, respectively. METHODS: We compared prospectively the Idylla platform with the results we obtained from parallel high-throughput next-generation sequencing, which is the current gold standard for mutational testing. Furthermore, we evaluated the benefits and disadvantages of the Idylla platform in clinical practice. Additionally, we reviewed all the published Idylla performance articles. RESULTS: There was an overall agreement of 100%, 94% and 94% between the next-generation panel and the Idylla BRAF, KRAS and EGFR mutation test. Two interesting discordant findings among 48 cases were observed and will be discussed together with the advantages and shortcoming of both techniques. CONCLUSION: Our observations demonstrate that the Idylla cartridge for the EGFR, KRAS and BRAF mutations is highly accurate, rapid and has a limited hands-on time compared with next-generation sequencing.

RNA-sequencing-based comparative analysis of human hepatic progenitor cells and their niche from alcoholic steatohepatitis livers.

Hepatic progenitor cells (HPCs) are small cells with a relative large oval nucleus and a scanty cytoplasm situated in the canals of Hering that express markers of (immature) hepatocytes and cholangiocytes. HPCs are present in large numbers in alcoholic steatohepatitis (ASH), one of the leading causes of chronic liver disease. To date, the mechanisms responsible for proliferation and differentiation of human HPCs are still poorly understood and the role of HPCs in ASH development is unknown. In this study, we aimed to characterise human HPCs and their interactions with other cells through comparison, on both protein and RNA level, of HPC-enriched cell populations from adult human liver tissue using different isolation methods. Fresh human liver tissue was collected from ASH explant livers and HPC-enriched cell populations were obtained via four different isolation methods: side population (SP), epithelial cell adhesion molecule (EpCAM) and trophoblast antigen 2 (TROP-2) membrane marker isolation and laser capture microdissection. Gene expression profiles of fluorescent-activated cell-sorted HPCs, whole liver extracts and laser microdissected HPC niches were determined by RNA-sequencing. Immunohistochemical evaluation of the isolated populations indicated the enrichment of HPCs in the SP, EpCAM+ and TROP-2+ cell populations. Pathway analysis of the transcription profiles of human HPCs showed an enrichment and activation of known HPC pathways like Wnt/ß-catenin, TWEAK and HGF. Integration of the HPC niche profile suggests autocrine signalling by HPCs (TNFα, PDGFB and VEGFA) as well as paracrine signalling from the surrounding niche cells including MIF and IGF-1. In addition, we identified IL-17 A signalling as a potentially novel pathway in HPC biology. In conclusion, we provide the first RNA-seq-based, comparative transcriptome analysis of isolated human HPCs from ASH patients and revealed active signalling between HPCs and their surrounding niche cells in ASH livers and suggest that HPCs can actively contribute to liver inflammation.

Mild hypothermia reduces activated caspase-3 up to 1 week after a focal cerebral ischemia induced by endothelin-1 in rats.

Hypothermia is a promising neuroprotective therapy that has been shown to reduce apoptosis after an ischemic insult. This study evaluated the effect of mild hypothermia on activated caspase-3 up to 1 week after the induction of a stroke. Endothelin-1 (Et-1) was used to elicit transient focal cerebral ischemia in rats. Twenty minutes after the ischemic insult, a state of mild hypothermia (33°C) was imposed for a duration of 2h. The functional outcome, infarct volume and activated caspase-3 immunoreactivity (IR) were assessed at 8, 24 and 72h, and one week after the insult. During the experiment the cerebral blood flow (CBF) was measured via Laser Doppler Flowmetry. Hypothermia improved the neurological outcome at all of the time points studied compared to the normothermic group, and was associated with a reduction in infarct volume. In both groups, activated caspase-3 IR peaked 24h after the Et-1 induced insult and hypothermia significantly reduced the number of apoptotic cells at 8h, 24h and 1 week after ischemia. Furthermore, the hypothermic treatment did not affect the CBF in the Et-1 model. These findings indicate that in the Et-1 model, hypothermia exerts a long lasting effect on stroke-induced apoptosis.

Proteomic analysis of global protein expression changes in the endothelin-1 rat model for cerebral ischemia: rescue effect of mild hypothermia.

Mild hypothermia is a promising neuroprotective therapy in stroke management. However, little is known about its effects on the global protein expression patterns in brain regions affected by ischemic stroke. We investigated protein expression changes associated with the neuroprotective effects of hypothermia via a functional proteomics approach through the analysis of the core (striatum) and the penumbra (cortex) after an ischemic insult in rats induced by endothelin-1 (Et-1). Functional outcome, infarct volume and related global protein expression changes were assessed 24h after the insult using two-dimensional difference gel electrophoresis. Mild hypothermia, induced 20 min after endothelin-1 infusion, improved the neurological outcome, reflected by a 36% reduction in infarct volume and a significantly better neurological deficit score. Hypothermia was typically associated with opposite protein expression changes inthe cortex to those induced by stroke under normothermic conditions, but not in the striatum. The main cellular processes rescued by hypothermia and potentially involved in the protection of the cortex are cellular assembly and organization, followed by cell signaling, thereby confirming that hypothermia is neuroprotective through multiple molecular and cellular pathways.

MALDI MS imaging as a tool for biomarker discovery: methodological challenges in a clinical setting.

MALDI MS imaging (MSI) is an analytical tool capable of providing spatial distribution and relative abundance of biomolecules directly in tissue. After 15 years of intense efforts to improve the acquisition and quality of molecular images, MSI has matured into an asset of the proteomic toolbox. The power of MSI lies in the ability to differentiate tissue regions that are not histologically distinct but are characterized by different MS profiles. Recently, MSI has been gaining momentum in biomedical research and has found applications in disease diagnosis and prognosis, biomarker discovery, and drug therapy. Although the technology holds great promise, MSI is still faced with a set of methodological challenges presented by the clinical setting. There is a growing awareness regarding this topic and efforts are being taken to develop clear and practical standards to overcome these challenges. This review presents an overview of MALDI MSI as a biomarker discovery tool and recent methodological progress in the field.

Experimental and clinical use of therapeutic hypothermia for ischemic stroke: opportunities and limitations.

Stroke remains a disease with a serious impact on quality of life but few effective treatments exist. There is an urgent need to develop and/or improve neuroprotective strategies to combat this. Many drugs proven to be neuroprotective in experimental models fail to improve patient outcome in a clinical setting. An emerging treatment, therapeutic hypothermia (TH), is a promising neuroprotective therapy in stroke management. Several studies with TH in experimental models and small clinical trials have shown beneficial effects. Despite this, implementation into the clinical setting is still lacking due to methodological considerations as well as hypothermia-related complications. This paper discusses the possible opportunities and limitations of the use of TH in animal models and the translation into the clinic.

Serial semiquantitative imaging of brain damage using micro-SPECT and micro-CT after endothelin-1-induced transient focal cerebral ischemia in rats.

UNLABELLED: In this study, we validated the use of (99m)Tc-hexamethylpropyleneamine oxime ((99m)Tc-HMPAO) micro-SPECT combined with micro-CT for semiquantification of the infarct size after an experimental stroke in rats and compared our observations with those obtained from histology. This imaging strategy was applied to measure the longitudinal effect of mild hypothermia on the progression of brain damage after stroke in rats. METHODS: The endothelin-1 model was used to elicit a transient focal cerebral ischemia in rats. This resulted in a reproducible insult in which the core is represented by the striatum and the penumbra by the cortex. Micro-SPECT and micro-CT images were taken at 1, 3, and 7 d after infusion of endothelin-1 and compared with those taken before the insult. After the last acquisition, rats were sacrificed and the infarct volume was determined via Nissl staining. The results obtained with micro-SPECT and micro-CT were compared with histology at the same time points. Mild hypothermia (33°C) was induced for 2 h, starting 20 min after the insult. RESULTS: Brain damage was estimated using micro-SPECT and micro-CT and was reproducible with minimal interobserver variability. Normothermic stroke rats had reduced (99m)Tc-HMPAO uptake at 1 and 3 d after the insult, whereas hypothermia improved damage after stroke. These findings corroborate with histology at the same time points. At 1 wk after the insult, no reduction of radioactive uptake was observed in any treatment group. CONCLUSION: Micro-SPECT and micro-CT allow quick and reproducible semiquantification of brain damage as an interesting alternative to histology to measure the extent of infarcted tissue in small animals after stroke.