Radioresistant glioma stem cells—Therapeutic obstacle or promising target?

Attempts to improve outcomes for patients with glioblastoma multiforme have focused largely on dose intensification and multimodal therapy and have been met with limited success. An alternative approach involves identifying and targeting the mechanisms responsible for tumour resistance. Recent data suggests that these high grade malignancies harbour a population of glioma stem cells that are both radioresistant and capable of initiating tumour regrowth. Furthermore, the radioresistant phenotype may be the consequence of constitutively upregulated and hyper-responsive cell cycle checkpoint pathways. This article considers whether these findings herald the dawn of a new era of effective, targeted therapy for this refractory tumour.     

Melanoma‐initiating cells: a compass needed

Most tumours contain a heterogeneous population of cancer cells, which harbour a range of genetic mutations and have probably undergone deregulated differentiation programmes that allow them to adapt to tumour microenvironments. Another explanation for tumour heterogeneity might be that the cells within a tumour are derived from tumour‐initiating cells through diverse differentiation programmes. Tumour‐initiating cells are thought to constitute one or more distinct subpopulations within a tumour and to drive tumour initiation, development and metastasis, as well as to be responsible for their recurrence after therapy. Recent studies have raised crucial questions about the nature, frequency and importance of melanoma‐initiating cells. Here, we discuss our current understanding of melanoma‐initiating cells and outline several approaches that the scientific community might consider to resolve the controversies surrounding these cells.     

Assessment of errors associated with harbour seal (Phoca vitulina) faecal sampling

Six harbour seals, ages 4–8 years, were held as pairs in a 10 times 20 times 2 m tank filled with sea water, and on 60 occasions were fed a meal of a specific species of fish or cephalopod of known size. The tank was drained periodically, and harbour seal faeces were collected on a 0.5 mm sieve. Number and size of otoliths and beaks found in faeces were determined. Fifty-eight percent of 670 fish and 37% of 36 cephalopods fed to harbour seals were represented by their otoliths or beaks in faeces. Estimated number of prey consumed was determined from the greatest number of left or right otoliths or upper or lower beaks collected in faeces. Estimated length ofprey was determined from measurements of otoliths and beaks recovered in the tank and relationships of otolith and beak measurements to prey length. Estimated number of fish eaten was not significantly different among pairs of harbour seals, but was different among species of fishes. Only 24–35% of fish species with small otoliths were represented in faeces, whereas more robust otoliths from other species were less apt to be completely dissolved. Estimated length of fishes was significantly less than lengths of fishes fed to harbour seals in 39 (76.5%) of 51 trials. Cephalopod beaks were not affected by passage through the harbour seal digestive tract. Amount of otolith dissolution was not related to species of fish; estimated fish length was underestimated by an average 27.5%. Although some (7.4%) of the otoliths were collected within 100 h after the fish were ingested, more than 90% were recovered within 24 h after the fish was eaten. Correction factors were developed which will allow researchers to estimate more reliably number and size of fish and cephalopod prey eaten by harbour seals.     

Augmented CD133 expression in distal margin correlates with poor prognosis in colorectal cancer

Pathological assessment of excised tumour and surgical margins in colorectal cancer (CRC) play crucial role in prognosis after surgery. Molecular assessment of margins could be more sensitive and informative than conventional histopathological analysis. Considering this view, we evaluated the distal surgical margins for expression of cancer stem cell (CSC) markers. Cellular and molecular assessment of normal, tumour and distal margin tissues were performed by flow cytometry, real‐time q‐PCR and immuno‐histochemical analysis for CRC patients after tumour excision. CRC patients were evaluated for expression of CSC markers in their normal, tumour and distal tissues. Flow cytometry assay revealed CD133 and CD44 enriched cells in distal margin and tumour compared to normal colorectal tissues, which was further confirmed by immunohistochemistry. Most importantly, immunohistochemistry also revealed the enrichment of CSC markers expression in pathologically negative distal margins. Patients with distal margin enriched for CD133 expression showed an increased recurrence rate and decreased disease‐free survival. This study proposes that although distal margin seems to be tumour free in conventional histopathological analysis, it could harbour cells enriched for CSC markers. Further CD133 could be a promising molecule to be used in molecular pathology for disease prognosis after surgery in CRC patients.     

Tumour escape mechanisms and their therapeutic implications in combination tumour therapy

Most tumours arise from a single normal cell through a sequential evolutionary process of mutation and selection. Tumours are initiated by escaping non‐immune surveillance, which includes defective DNA repair, epigenetic gene alternation, resistance to apoptosis and loss of intercellular contact inhibition. Tumour cells harbour mutations in a number of critical genes that provide selective advantages at various stages during the evolution of the tumour. The tumour cells that circumvent the tumour suppressor mechanisms of the non‐immune surveillance process are edited by the immune system, resulting in the selection of a resistant tumour variant. The selection of the tumour cell is further shaped by its interactions with cells and other factors in its microenvironment. Tumour evolution is thought to adhere to Darwinian principles by escaping both non‐immune (intrinsic) and immune (extrinsic) responses against self‐altered tumour cells. At end‐stage, tumours have escaped both non‐immune and immune surveillance with increased threshold of apoptosis. Combination therapy has been proposed, by exploring the non‐immune and immune suppressive nature of the tumour, and has been found to have a therapeutic efficiency on tumour regression as compared with monotherapies. The combination of immunotherapy and other different modalities, especially vaccines, with conventional anticancer therapies with optimized dosage and scheduling can offer synergistic antitumour effects. Here, we focus on the mechanism of tumour evolution and its implication in combination therapy.     

Four separate regions on chromosome 17 show loss of heterozygosity in familial breast carcinomas

Two genes predisposing females to autosomal dominant breast cancer are located on chromosome 17. Mutations in the p53-gene on the short arm have been shown to predispose females to early onset breast cancer in families with the rare Li-Fraumeni syndrome. Another locus on 17q (BRCA1), was found to be linked to the disease in a subset of families with breast cancer. In order to determine the involvement of tumour suppressor genes at these loci in tumour development, we studied allele losses for markers on chromosome 17 in 78 familial breast carcinomas. The analysis used six polymorphic DNA markers, three on each arm. We found support for at least four separate regions displaying allele losses on chromosome 17: the p53-region, the distal part of 17p, the BRCA1 region and the distal part of 17q. The frequency of allele losses on distal 17p (16%) is low in these familial tumours compared with the previously reported incidence in sporadic tumours (>50%), whereas the frequency of losses at the p53 locus and on 17q was similar to sporadic tumours (5%–40%). These data suggest that several regions on chromosomal 17 can harbour tumour suppressor genes involved in tumour development of familial breast cancer.     

Transmissible cancer in Tasmanian devils: localized lineage replacement and host population response

Tasmanian devil facial tumour disease (DFTD) is a clonally transmissible cancer threatening the Tasmanian devil (Sarcophilus harrisii) with extinction. Live cancer cells are the infectious agent, transmitted to new hosts when individuals bite each other. Over the 18 years since DFTD was first observed, distinct genetic and karyotypic sublineages have evolved. In this longitudinal study, we investigate the associations between tumour karyotype, epidemic patterns and host demographic response to the disease. Reduced host population effects and low DFTD infection rates were associated with high prevalence of tetraploid tumours. Subsequent replacement by a diploid variant of DFTD coincided with a rapid increase in disease prevalence, population decline and reduced mean age of the population. Our results suggest a role for tumour genetics in DFTD transmission dynamics and epidemic outcome. Future research, for this and other highly pathogenic emerging infectious diseases, should focus on understanding the evolution of host and pathogen genotypes, their effects on susceptibility and tolerance to infection, and their implications for designing novel genetic management strategies. This study provides evidence for a rapid localized lineage replacement occurring within a transmissible cancer epidemic and highlights the possibility that distinct DFTD genetic lineages may harbour traits that influence pathogen fitness.     

Gamma Knife(R) radiosurgery for recurrent intracranial olfactory neuroblastoma (esthesioneuroblastoma): a case report

ABSTRACT: BACKGROUND: We report the use of salvage radiosurgery to manage an aggressive olfactory neuroblastoma(esthesioneuroblastoma) with multiple recurrences and intracranial extension. CASE PRESENTATION: A 43-year-old Caucasian woman presented 11 years ago with progressive nasal blockage andheadaches. A necrotic polyp originating in her left middle meatus and extending to theethmoid air cells and cribriform plate (Kadish stage C) was radically resected via acraniofacial approach. Four years later, a local recurrence extending into her left cavernoussinus was identified and deemed inoperable. She received vincristine, ifosfamide,doxorubicin and etoposide chemotherapy (with minimal benefit) and external beamradiotherapy (60Gy in 30 fractions) to her skull base. Two years later, tumour extension inher left neck was treated with radical radiotherapy. She developed visual disturbances in herleft eye, which progressed to blindness in the next two years. Having exhaustedchemoradiotherapy, the left cavernous sinus esthesioneuroblastoma was treated with GammaKnife? radiosurgery 2 years ago (20Gy at 50% isodose, tumour volume 7.5cm3). At oneyear, there was dramatic reduction in the tumour and no new symptoms; however, there werenew tumour foci (in her left frontal lobe and above her right orbital apex). These were againtreated with radiosurgery (20Gy at 50% isodose, total tumour volume 0.67cm3). Repeatimaging at six months showed no further disease progression. CONCLUSION: Whilst rare, olfactory neuroblastoma (esthesioneuroblastoma) can present managementchallenges and Gamma Knife(R) radiosurgery may prove a useful strategy in controllingintracranial spread.     

The adenomatous polyposis coli‐associated exchange factors Asef and Asef2 are required for adenoma formation in ApcMin/+mice

Sporadic and familial colorectal tumours usually harbour biallelic adenomatous polyposis coli (APC)‐associated mutations that result in constitutive activation of Wnt signalling. Furthermore, APC activates Asef and Asef2, which are guanine‐nucleotide exchange factors specific for Rac1 and Cdc42. Here, we show that Asef and Asef2 expression is aberrantly enhanced in intestinal adenomas and tumours. We also show that deficiency of either Asef or Asef2 significantly reduces the number and size of adenomas in Apc^Min/+ mice, which are heterozygous for an APC mutation and spontaneously develop adenomas in the intestine. We observed that the APC–Asef/Asef2 complex induces c‐Jun amino‐terminal kinase‐mediated transactivation of matrix metalloproteinase 9, and is required for the invasive activity of colorectal tumour cells. Furthermore, we show that Asef and Asef2 are required for tumour angiogenesis. These results suggest that Asef and Asef2 have a crucial role in intestinal adenoma formation and tumour progression, and might be promising molecular targets for the treatement of colorectal tumours.     

Carcinoid tumour secreting dopa.

A middle aged woman referred for an abdominal mass was found to have large amounts of dopa (3-4-dihydroxyphenylalanine) metabolites in her urine. At operation a tumour affecting almost the entire left lobe of the liver was removed. Histologically the tumour was a metastatic carcinoid. After operation the excretion of dopa metabolites fell substantially, confirming that the tumour was the source. Apparently, owing to an enzyme defect the tumour had been unable to decarboxylate dopa. These findings are further evidence of a neural origin for the endocrine system of the gut.     

The cytotoxic activity of the bacteriophage lambda-holin protein reduces tumour growth rates in mammary cancer cell xenograft models

BACKGROUND: The potential use of gene therapy for cancer treatment is being intensively studied. One approach utilises the expression of genes encoding cytotoxic proteins. Such proteins can affect cellular viability, for example by inhibiting the translation machinery or disturbing membrane integrity. The bacteriophage Lambda (lambda)-holin protein is known to form a lesion in the cytoplasmic membrane of E. coli, triggering bacterial cell lysis and thereby enabling the release of new bacteriophage particles. The aim of this study was to evaluate whether the lambda-holin protein has a cytotoxic impact on eukaryotic cells and whether it holds potential as a new therapeutic protein for cancer gene therapy. METHODS: To explore this possibility, stably transfected human cell lines were established that harbour a tetracycline (Tet)-inducible system for controlled expression of the lambda-holin gene. The effect of the lambda-holin protein on eukaryotic cells was studied in vitro by applying several viability assays. We also investigated the effect of lambda-holin gene expression in vivo using a human breast cancer cell tumour xenograft as well as a syngeneic mammary adenocarcinoma mouse model. RESULTS: The lambda-holin-encoding gene was inducibly expressed in eukaryotic cells in vitro. Expression led to a substantial reduction of cell viability of more than 98%. In mouse models, lambda-holin-expressing tumour cell xenografts revealed significantly reduced growth rates in comparison to xenografts not expressing the lambda-holin gene. CONCLUSIONS: The lambda-holin protein is cytotoxic for eukaryotic cells in vitro and inhibits tumour growth in vivo suggesting potential therapeutic use in cancer gene therapy.     

Hydrodynamic perception in true seals (Phocidae) and eared seals (Otariidae)

Pinnipeds, that is true seals (Phocidae), eared seals (Otariidae), and walruses (Odobenidae), possess highly developed vibrissal systems for mechanoreception. They can use their vibrissae to detect and discriminate objects by direct touch. At least in Phocidae and Otariidae, the vibrissae can also be used to detect and analyse water movements. Here, we review what is known about this ability, known as hydrodynamic perception, in pinnipeds. Hydrodynamic perception in pinnipeds developed convergently to the hydrodynamic perception with the lateral line system in fish and the sensory hairs in crustaceans. So far two species of pinnipeds, the harbour seal (Phoca vitulina) representing the Phocidae and the California sea lion (Zalophus californianus) representing the Otariidae, have been studied for their ability to detect local water movements (dipole stimuli) and to follow hydrodynamic trails, that is the water movements left behind by objects that have passed by at an earlier point in time. Both species are highly sensitive to dipole stimuli and can follow hydrodynamic trails accurately. In the individuals tested, California sea lions were clearly more sensitive to dipole stimuli than harbour seals, and harbour seals showed a superior trail following ability as compared to California sea lions. Harbour seals have also been shown to derive additional information from hydrodynamic trails, such as motion direction, size and shape of the object that caused the trail (California sea lions have not yet been tested). The peculiar undulated shape of the harbour seals’ vibrissae appears to play a crucial role in trail following, as it suppresses self-generated noise while the animal is swimming.     

Neurogenic differentiation of amniotic fluid stem cells

In 2003, human amniotic fluid has been shown to contain stem cells expressing Oct-4, a marker for pluripotency. This finding initiated a rapidly growing and very promising new stem cell research field. Since then, amniotic fluid stem (AFS) cells have been demonstrated to harbour the potential to differentiate into any of the three germ layers and to form three-dimensional aggregates, so-called embryoid bodies, known as the principal step in the differentiation of pluripotent stem cells. Marker selection and minimal dilution approaches allow the establishment of monoclonal AFS cell lineages with high proliferation potential. AFS cells have a lower risk for tumour development and do not raise the ethical issues of embryonic stem cells. Compared to induced pluripotent stem cells, AFS cells do not need exogenic treatment to induce pluripotency, are chromosomal stable and do not harbour the epigenetic memory and accumulated somatic mutations of specific differentiated source cells. Compared to adult stem cells, AFS can be grown in larger quantities and show higher differentiation potential. Accordingly, in the recent past, AFS became increasingly accepted as an optimal tool for basic research and probably also for specific cell-based therapies. Here, we review the current knowledge on the neurogenic differentiation potential of AFS cells.     

An unusual case of recurrent left atrial myxoma.

In a patient who had a calcified left atrial myxoma resected, recurrence developed 31 months later. Although complete radical resection of the recurrent tumour presented a special problem, the patient survived the second operation. The tumour recurred again and the patient had two episodes of cerebral embolism 1 1/2 and 2 years later, respectively, and died 3 1/2 years after the second operation. The erythrocyte sedimentation rate correlated with the size of the tumor, and the recurrent tumour seemed to grow more rapidly than the primary tumour. Experience with this case and a review of the nine reported cases of recurrent left atrial myxoma suggest that a radical approach is necessary at the primary operation.     

An assessment of population size and distribution of harbour seals in the Republic of Ireland during the moult season in August 2003

The status of Ireland's harbour seal Phoca vitulina vitulina population is poorly understood. The most recent national population estimate dates back to the breeding season in 1978 and did not cover the entire coastline. Reliable up-to-date information on the abundance and distribution of harbour seals in Ireland is necessary to assess the conservation status of the species and for the effective identification, management and monitoring of special areas of conservation required for harbour seals under the EU Habitats Directive. To provide comprehensive current information on Ireland's harbour seal population, a geographically extensive survey was conducted along the coastline of the Republic of Ireland during the species' annual moult in August 2003. This complemented a similar survey of Northern Ireland, which was conducted in 2002. Using thermal imaging technology, haul-out groups of harbour seals and grey seals Halichoerus grypus were identified from the air, aerial-counts were obtained and compared with simultaneous ground-count data from selected sites. Harbour seal distribution recorded during the 2003 moult season was concentrated in the south-west, west and north-west of the country. This national survey yielded a minimum population estimate for the Republic of Ireland of 2905 harbour seals, delivering an effective baseline for current and future population research.     

Polychaete assemblages and sediment pollution in a harbour with two opposing entrances

The harbour at Ceuta is one of the most important harbours in the Strait of Gibraltar. The sediments are moderately polluted with organic matter and heavy metals but the harbour has two opposing entrances and a connecting channel which increases water renewal and dissolved oxygen across the harbour. For these special conditions, the value of the soft bottom polychaete community as a bioindicator, and possible advantages of the presence of two harbour entrances on biotic assemblages, were studied. Twenty-one stations were selected, and 27 variables were measured in the sediment of each station. The polychaete species richness and Shannon diversity values were similar inside and outside the harbour. Nevertheless, the Pielou evenness index was significantly higher in the external stations due to high densities of some species of polychaetes such as Pseudomalacoceros tridentata and Capitella capitata inside the harbour. The multivariate approach based on polychaete species composition was much more sensitive than univariate analyses at discriminating between internal and external stations. The pollution gradient and granulometric parameters were the main factors affecting polychaete distribution. Polychaete species richness and diversity in sediments inside Ceuta harbour were higher than in conventional harbours due to the positive effects of increased water renewal. These results should be taken into consideration in design, construction and remodelling of future harbours.Communicated by: H.-D. Franke     

Diagnosis of left atrial myxoma by echocardiography

The diagnostic use of ultrasound was demonstrated in two patients with left atrial myxoma. In both cases the decision to proceed with confirmatory angiography and ultimate removal of the tumour was initiated by the echocardiographic findings. The echocardiogram proved to be a simple non-invasive technique for the diagnosis of left atrial mass.     

Rookery through rehabilitation: Microbial community assembly in newborn harbour seals after maternal separation

Microbial community assembly remains largely unexplored in marine mammals, despite its potential importance for conservation and management. Here, neonatal microbiota assembly was studied in harbour seals (Phoca vitulina richardii) at a rehabilitation facility soon after maternal separation, through weaning, to the time of release back to their native environment. We found that the gingival and rectal communities of rehabilitated harbour seals were distinct from the microbiotas of formula and pool water, and became increasingly diverse and dissimilar over time, ultimately resembling the gingival and rectal communities of local wild harbour seals. Harbour seal microbiota assembly was compared to that of human infants, revealing the rapid emergence of host specificity and evidence of phylosymbiosis even though these harbour seals had been raised by humans. Early life prophylactic antibiotics were associated with changes in the composition of the harbour seal gingival and rectal communities and surprisingly, with transient increases in alpha diversity, perhaps because of microbiota sharing during close cohabitation with other harbour seals. Antibiotic-associated effects dissipated over time. These results suggest that while early life maternal contact may provide seeding for microbial assembly, co-housing of conspecifics during rehabilitation may help neonatal mammals achieve a healthy host-specific microbiota with features of resilience.     

How does the metabolism of tumour cells differ from that of normal cells

Tumour cells thrive in environments that would be hostile to their normal cell counterparts. Survival depends on the selection of cell lines that harbour modifications of both, gene regulation that shifts the balance between the cell cycle and apoptosis and those that involve the plasticity of the metabolic machinery. With regards to metabolism, the selected phenotypes usually display enhanced anaerobic glycolysis even in the presence of oxygen, the so-called Warburg effect, and anabolic pathways that provide precursors for the synthesis of lipids, proteins and DNA. The review will discuss the original ideas of Otto Warburg and how they initially led to the notion that mitochondria of tumour cells were dysfunctional. Data will be presented to show that not only the organelles are viable and respiring, but that they are key players in tumorigenesis and metastasis. Likewise, interconnecting pathways that stand out in the tumour phenotype and that require intact mitochondria such as glutaminolysis will be addressed. Furthermore, comments will be made as to how the peculiarities of the biochemistry of tumour cells renders them amenable to new forms of treatment by highlighting possible targets for inhibitors. In this respect, a case study describing the effect of a metabolite analogue, the alkylating agent 3BP (3-bromopyruvate), on glycolytic enzyme targets will be presented.     

Maximum likelihood population size estimation of harbour seals in the Dutch Wadden Sea based on a mark–recapture experiment

1. The harbour seal population Phoca vitulina in the entire Wadden Sea was severely depleted due to a virus-epizootic during 1988. A comprehensive study on the population biology and activity patterns was subsequently initiated to design a management and conservation plan. The main objective of this study was to estimate harbour seal abundance in the different regions of the Wadden Sea.
2. We investigated the potential of a mark–recapture experiment using VHF radio-tags in combination with repeated aerial surveys to estimate the number of harbour seals in the Dutch part of the Wadden Sea. The number of harbour seals hauled-out and the presence of any radio-tagged seals was monitored during seven aerial surveys of all known haul-out sites in the Dutch Wadden Sea over the 1994 breeding season.
3. A maximum likelihood (ML) estimator was developed to infer the rate of tag-loss and the size of the local prepupping population.
4. The ML estimate of the number of harbour seals in the Dutch Wadden Sea was 1536 (95% confidence limits were 1225 and 2200). The corresponding maximum proportion of seals hauled-out was 68%.
5. The use of VHF radio-tags which can be monitored from the air provides a way of correcting aerial survey counts for the proportion of harbour seals hauled-out during the surveys. Since haul-out behaviour may be influenced by local conditions, such as exposure time of sand banks, we recommend this technique be repeated in other areas of the Wadden Sea rather than using the estimates from the current study in other areas.