TaqMan genotyping with real-time PCR is a reliable method for single nucleotide polymorphism detection, which is done by probes. These oligonucleotides should be short enough to avoid mismatch hybridization, as well as having 5–10°C higher melting temperature than the primers of real-time PCR reaction. One approach for these qualities is to conjugate the probe with minor groove binder (MGB). Having no access to MGB probes, we searched for an alternative. In the current study, we used Zip Nucleic Acids (ZNA) as probes to increase its stability and melting temperature. Our aim was to genotype the -265 T/C changes of Apolipoprotein A-2 gene. We set up the real-time PCR reaction with ZNA probes, and by repeating the reactions, we confirmed the reliability of this new approach. It is now recommended to use ZNA probes, as an alternative to MGB probes, to increase the probe Tm value and its binding to target DNA. A single-nucleotide polymorphism (SNP) is the variation of a single nucleotide in DNA sequence between two or more alleles. SNP genotyping is the study of such variations across the genome. It can be applied with different methods. Among them, TaqMan genotyping assay with the real-time PCR is a gold standard for the analysis of a limited number of SNPs in large sample collections, specifically when the site of an SNP is not detectable with a restriction enzyme. It allows the detection of both SNP alleles in a single reaction tube ,2.In our study, the desired SNP was not detectable with any restriction enzyme. This SNP is in the promoter region of Apolipoprotein A-2 gene . Therefore, we decided to carry out the TaqMan genotyping assay with real-time PCR. In order to have the most reliable result, we had to detect both alleles simultaneously. Thus we applied two different probes in each reaction. The aim of the study was to analyze this SNP among 800 Diabetes mellitus type 2 patients.Technically, in TaqMan genotyping, each probe is a linear oligonucleotide with a fluorogenic dye attached to the 5′ end and a quencher molecule to the 3′ end. The close proximity of the reporter and quencher prevents the fluorescence emission. In each PCR cycle, after primer and probe annealing, the Taq polymerase attaches to the 3′ end of the primer and synthesize the new complementary strand. The Taq polymerase used in TaqMan genotyping has an additional 5′ to 3′ exonuclease activity. This causes the degradation of probe, and thus the release of the reporter dye fluorescence ,3.The point is the annealing temperature of probes and primers. These oligonucleotides should be designed in a way that in reaction, first the probes, and then the primers should be hybridized with the DNA. So the melting temperature (Tm) of probes should be more than primers. Simply, it means the probes should be longer than primers. However, longer the probes increase the chance of mismatch formation between probes and target DNA. Longer probes are less sensitive to mismatch discrimination. This will cause false positive results, especially when the difference between 2 or more probes is only a single nucleotide. Therefore, designing longer probes is not the right solution .In order to increase the Tm value of the probes, without increasing the probe length, one way is to use MGB (Minor Groove Binder). Attaching MGB to the 3′ end of TaqMan probes increases the stability of hybridization between the probe and target DNA. This results in the increase of probe melting temperature. By increasing the Tm value, the design of shorter probes is now possible. Shorter probes make them more specific to determine a single nucleotide difference. By using the shorter probes with higher Tm values, there would be no risk of mismatch formation between a false probe and the target DNA molecule .In Iran, we do not have access to TaqMan probes, since they are only produced by Applied Biosystems, which is an American company. We had to look for an alternative way to increase the Tm value of probes. We searched many companies which deals with oligonucleotide synthesis, and finally found that a German company, Metabion Inc., has just introduced a new method to increase the Tm value, called ZNA.Zip nucleic acid (ZNA) is generally an oligonucleotide which is conjugated with a cationic compound, named spermine. The role of spermine is to increase the affinity between the oligonucleotide and the target nucleic acid molecule. This results in decreasing the electrostatic repulsion between these two negatively charged molecules . The melIn our study, we used the ZNA probes to genotype the -265 T/C changes of Apo A-2 gene. We made use of ZNA unique features for increasing the Tm value of our probes, and thus to avoid having a mismatch between the false probe and the target DNA. The probe sequences are: ApoA2-Ca: 5′-FAM- TTGGACTTGAGTGCAACA-BHQ1-3′; and ApoA2-Ta: 5′-JOE- CTTGGACTTGAATGCAACA-BHQ1-3′. The specific nucleotide for detection of T > C change is underlined. Without the spermine, the Tm of probes is 47.5°C and 49.8°C respectively; calculated by Nearest Neighbor method . By usinPCR was performed in a 10 μL final volume. The reaction mixture contained 5 μL TaqMan Universal Master Mix II (Applied Biosystems), 200 nmol/L of both probes, 900 nmol/L of both forward and reverse primers , and 25 ng genomic DNA. The thermal cycler was run with 40 cycles at 95°C for 15 s and 60°C for 1 minute. Allelic discrimination was performed at the end of each cycle and on the post-PCR product. The reaction was run by StepOne Real-Time PCR System (Applied Biosystems).Before studying patient samples, we repeat the test on our reference DNA samples in triple times, whether in the same or different runs to ensure the reliability of our PCR setup. Moreover, we randomly chose some patient samples DNA and repeat the test on them. All this repetitions lead to the same results. Figure Zip Nucleic Acids are new in the field of oligonucleotide synthesis. Recently, two new researches are reported, which have used this type of oligonucleotides. Moreau et al. used ZNA as primers. They reported that these primers can improve the yield of cDNA synthesis, especially for the genes expressed at low levels . In the In the current study, we also set up our experiment with ZNA probes. We run real-time PCR of 800 samples, and we repeat some of these tests. In all cases, we reached the same results. Therefore, we confirmed that this type of oligonuclotide is reliable to be used as real-time PCR probe. This study is another proof to show the accuracy of Zip Nucleic Acids.While having no access to MGB-conjugated TaqMan probe, we decided to use an alternative, called ZNA to increase the melting temperature of the probes. Our data suggest that these probes can provide an effective and reliable alternative to MGB for SNP genotyping.


Clostridium has shown great potential in penetration and colonization of the hypoxic and necrotic areas of the tumour microenvironment, causing significant oncolysis as well as enabling the delivery of therapeutics directly to the tumour in situ. Various strategies utilizing Clostridium are currently being investigated, and represent a novel area of emerging cancer therapy. This review provides an update review of tumour microenvironment as well as summary of the progresses and current status of Clostridial spore-based cancer therapies.Solid tumour accounts for 90% of all cancers. The current treatment approach for most solid tumours is surgery, however it is limited to early stage tumours. Other treatment options such as chemotherapy and radiotherapy are non-selective, thus causing damage to both healthy and cancerous tissue. Past research has focused on understanding tumour cells themselves, and conventional wisdom has aimed at targeting these cells directly. Recent research has shifted towards understanding the tumour microenvironment and it's differences from that of healthy cells/tissues in the body and then to exploit these differences for treatmeat of the tumour. One such approach is utilizing anaerobic bacteria. Several strains of bacteria have been shown to selectively colonize in solid tumours, making them valuable tools for selective tumour targeting and destruction. Amongst them, the anaerobic  Cancer is currently a major cause of morbidity and mortality internationally and poses a significant burden on both patients and their families and the healthcare system as a whole. Solid tumours, in particular, account for 90% of all cancers. A solid tumour is composed of a complex mix of tumour cells and nontumour cells, including supporting stromal and infiltrating blood cells, immune cells, and various molecules in proximity to these cells. This collection of cells and their metabolism is referred to as the tumour microenvironment. This unique tumour milieu not only allows for growth and metastasis, but additionally aids in the resistance of cancer cells to current chemotherapy and radiotherapy, thereby hindering their success. Consequently, there is an urgent demand for more suitable and effective treatment options for those suffering from solid tumours.Clostridium-spore-based bacterial therapy is one such a novel strategy.Research in the past has typically focused on understanding the molecular and genetic aspects of cancer cells, which led to treatment options geared towards killing the cancer cells themselves. Recent advances in the field of oncology have lead to a greater understanding of tumour pathology. Currently, a greater research emphasis has been placed on comprehending the unique environment of solid tumours, referred to as the tumour microenvironment. Developments in this field have lead to the possibility of pursuing methods of treatment which serve to control this milieu in such a way that will enable us to manage the growth and metastasis of the tumour itself. This has generated the potential for new therapeutic targets and treatment options for cancer patients.  Growth and progression of tumour cells requires an increased supply of both oxygen and nutrients. For this the formation of a new vascular network is required to meet these demands . Under r The key mediator of angiogenesis is the glycoprotein vascular endothelial growth factor (VEGF), which stimulates blood vessel growth by acting on endothelial cells . Additio Acute and chronic lack of blood supply resulting in the absence of oxygen and known as hypoxia is also a definitive characteristic of the tumour micro milieu and is caused by inefficient blood supply to cancerous cells. The hypoxic environment poses a challenge to traditional cancer treatment options in that this oxygen-depleted environment makes the solid tumour cells less susceptible to killing with chemotherapy and radiation, less genetically stable (thus more difficult to predict its response to treatment), and resistant to apoptosis . Since tα) plays a unique role in the tumour environment where it permits cellular communication and is capable of promoting both growth and necrosis. TNF-α acts in conjunction with many other molecules within the cell to perform these tasks. For instance, tumour necrosis is carried out first by endothelial cell apoptosis caused by deactivation of the integrin avb3 and disruption of the interface with the extracellular matrix (ECM), followed by T-cell activation to remove remaining tumour cells . COBALT is a proposed method of cancer treatment which has been relatively successful thus far. In this type of therapy, a bacterium is engineered to exhibit antitumour properties, such as proteolytic enzymes, which is then administered in conjunction with other known cancer therapies to work synergistically and improve oncolysis. A new strain of novyi-NT . Systemier cells . In an aC-novyi-NT has demonstrated that some of its oncolytic capabilities are due to the presence of several lipid degrading enzymes, which are highly expressed when C. novyi-NT is colonised in tumours [C. novyi-NT to release liposomal drugs within the tumour. It was hypothesised that since this species lyses red blood cells, its ability to disrupt membranes might be able to increase the release of liposome-encapsulated drugs directly at the site of the tumour. A form of doxorubicin incorporated within a liposome called Doxil was utilised, and promising results were obtained. Systemic injection of C. novyi-NT spores in conjunction with Doxil completely eliminated tumours in two models. Further analysis of protein fractions of the culture medium, as well as mass spectroscopy analysis, revealed that lipase was indeed the protein product secreted by C. novyi-NT which increased the effectiveness of the liposome-encapsulated drug. The significance of this study is that further research can be performed using other liposome encapsulated drugs with specific targeting and release within the tumour microenvironment [ Genome analysis of  tumours . In 2006 tumours , exploitironment , 48.Clostridium-directed therapies to be developed as a viable treatment option for cancer patients. Clostridium species which are easily transformable, such as C. acetobutylicum, do not typically have high colonisation efficiencies, whereas species which typically colonise well in tumour cells, such as C. sporogenes, C. oncolyticum, and C. novyi-NT are not easily transformed. However, recent advancements in genetic engineering technologies have allowed for improvements in this area. Using a new method of conjugative transfer from E. coli, Theys et al. were able to successfully transform plasmid vectors at higher frequencies into these strains of Clostridium, opening many new doors in Clostridium-directed cancer therapies [Clostridium can be successfully implemented in current cancer therapies, but research efforts continue and advances are being made regularly in this field. Although theoretically very promising, there are still some obstacles that must be overcome in order for herapies . KeepingClostridium-based cancer therapy is a promising approach for the treatment of solid tumours. Recently, understanding the solid tumour microenvironment and its influence on cancer therapy has profoundly changed our thinking about cancer therapy. We have realised that the unique solid tumour micro milieu has been one of the greatest hindrances thus far in successful cancer treatment. The utilisation of anaerobic Clostridium species allows for a targeted and curative treatment by destroying tumour microenvironment first, creating opportunity for combinational therapies. In addition, numerous strategies involving the administration of Clostridium spores to selectively deliver cancer therapeutics directly to the site of the solid tumour are currently being developed, and in many cases, promising oncolytic capabilities have been demonstrated. However, successful implementation of this mode of therapy in clinical trials relies on developmenting and manufacturing a Clostridium species to have both high colonisation efficiency and expression and excretion of sufficient high levels of the therapeutic proteins. Improving genetic engineering methods to genetically modify bacteria and modulation of gene expression to yield maximum protein secretion are areas which may enhance this field. Clostridium-based cancer therapies are one of the most novel and promising methods of cancer treatment currently being researched. The ability of these Clostridium based modalities to selectively target the microenvironment has provided a firm foundation for which to build towards efficient, safe and effective cancer treatments for the future and to improve the prognosis and treatment of so many individual patients suffering from solid tumours, relieving the burdens of patients, their families and the healthcare systems.


The following line was omitted from the Funding statement: "Industry Canada MNI center of excellence in commercialization and research grant awarded to AD and AS."


Mouse colorectal cancer (CRC) models generated by orthotopic microinjection of human CRC cell lines reproduce the pattern of lymphatic, haematological and transcoelomic spread but generate low metastatic efficiency. Our aim was to develop a new strategy that could increase the metastatic efficiency of these models. We used subcutaneous implantation of the human CRC cell lines HCT116 or SW48 prior to their orthotopic microinjection in the cecum of nude mice (SC+ORT). This subcutaneous preconditioning significantly enhanced metastatic dissemination. In the HCT116 model it increased the number and size of metastatic foci in lymph nodes, lung, liver and peritoneum, whereas, in the SW48 model, it induced a shift from non-metastatic to metastatic. In both models the number of apoptotic bodies in the primary tumour in the SC+ORT group was significantly reduced compared with that in the direct orthotopic injection (ORT) group. Moreover, in HCT116 tumours the number of keratin-positive tumour buddings and single epithelial cells increased at the invasion front in SC+ORT mice. In the SW48 tumour model, we observed a trend towards a higher number of tumour buds and single cells in the SC+ORT group but this did not reach statistical significance. At a molecular level, the enhanced metastatic efficiency observed in the HCT116 SC+ORT model was associated with an increase in AKT activation, VEGF-A overexpression and downregulation of β1 integrin in primary tumour tissue, whereas, in SW48 SC+ORT mice, the level of expression of these proteins remained unchanged. In summary, subcutaneous preconditioning increased the metastatic dissemination of both orthotopic CRC models by increasing tumour cell survival and invasion at the tumour invasion front. This approach could be useful to simultaneously study the mechanisms of metastases and to evaluate anti-metastatic drugs against CRC. When metastases are achieved, only microfoci are observed, limiting the analysis and clinical translation of the findings  cells that disseminate to all clinically relevant sites  . Most ofOur aim was to improve this orthotopic model by performing a prior single subcutaneous passage of HCT116 or SW48 CRC cells. We hypothesized that subcutaneous (SC) preconditioning before orthotopic microinjection would increase their metastatic efficiency. We also expected that the generated models would be useful to study the mechanisms of metastases and the preclinical development of novel anti-metastatic drugs.3) and ORT (1275±299 mm3) groups for HCT116, or between the SC+ORT (940.5±57.5 mm3) and ORT (940 mm3) groups for SW48. The SC+ORT and ORT groups in HCT116 and SW48 mice both developed undifferentiated tumours, with 40–70% necrosis and a high degree of vascular invasion. No significant differences were observed in mouse survival between the SC+ORT and the ORT groups for the HCT116  or SW48  models.There were no differences in the percentage of tumour engraftment between groups that underwent implantation of the human CRC cell lines HCT116 or SW48 (SC preconditioning) prior to their orthotopic microinjection (ORT) in the cecum of nude mice (SC+ORT) and ORT groups in the HCT116  or SW48  models. Moreover, we did not observe significant differences in primary tumour volume at the end of the experiment between the SC+ORT  are attributable to metastatic disease. Many patients present with metastatic, late-stage CRC at the time of diagnosis, and this can prove difficult to treat. However, current mouse models of CRC, generated by orthotopic microinjection of human CRC cell lines, rarely reproduce advanced stages of the disease and generate low metastatic efficiency. Thus, their use for understanding the mechanisms underlying metastasis and testing the efficacy of potential anti-metastatic therapies is limited.ResultsIn this study, the authors show that subcutaneous preconditioning prior to orthotopic injection of the HCT116 or SW48 CRC cell line in the cecum of nude (immunodeficient) mice generates models with enhanced metastatic efficiency in all clinically relevant sites: lymph nodes, liver, lung and peritoneum. Most importantly, macroscopic metastases in the liver, detected after injection of the HCT116 cell line, compromise liver function and lead to mouse death, recapitulating the progression of CRC in humans. These findings suggest that subcutaneous preconditioning induces a reduction in the rate of tumour cell death within primary tumours and an increased invasion at the tumour front, possibly explaining the increased dissemination and aggressiveness observed in the resulting models.Implications and future directionsin vivo models generated using this new approach could facilitate the study of the mechanisms of tumour invasion and metastatic dissemination and be used in the preclinical evaluation of anti-tumour and anti-metastatic compounds.This study provides a novel procedure that increases the metastatic efficiency of current CRC mouse models and reproduces the metastatic dissemination observed in humans. Given that metastatic rate is enhanced in two independent CRC models using this procedure, it is likely that subcutaneous preconditioning could improve the metastatic efficiency in other cancer models. The P<0.05) higher in the SC+ORT than in the ORT group. No significant differences between groups were recorded regarding the number of mice affected by liver or lymph node metastases, although the number and size of metastases were increased in these organs (see below). The direct orthotopic injection of the SW48 cell line (SW48 ORT) did not generate metastases in any of the injected animals. However, the SW48 SC+ORT group developed multiple metastases in the lymph nodes, lung and peritoneum. Thus, HCT116 SC+ORT mice had an enhanced metastatic rate and maintained the organo-tropism observed in the ORT group. Moreover, SC preconditioning switched the non-metastatic SW48 cell line to metastatic: SC+ORT SW48 mice developed metastases in most of the expected sites for CRC .Whereas no differences were observed in primary tumour growth or mouse survival, the number of HCT116 mice bearing lung metastases was significantly (P=0.003). HCT116 SC+ORT mice also developed significantly (P=0.020) larger visible metastases (790.7×105±148.1×105 μm2) than ORT mice (265.3×105±38.8×105 μm2)   and inva105 μm2) ; Table 1n=135) group was significantly (P≤0.01) higher than in the ORT group (n=18), which showed only micro- and macro-metastases, but no visible metastases. In the SC+ORT group, we identified 13 single visible metastases with an average size of 269.1×105±77.5×105 μm2 (5±6.7×105 μm2 in the SC+ORT group. The number of microfoci in this group (n=109) was significantly (P<0.01) higher than in the ORT group (n=18). Although mouse survival did not differ between groups, the two mice that developed massive liver metastases in the SC+ORT group had a shorter survival time (59±5 days) than the remaining animals (69±5 days), suggesting that liver metastases, rather than primary tumour growth, induced death in these mice.We also observed an enhanced efficiency of liver metastases in HCT116 SC+ORT mice as compared with the ORT group ; Table 1×105 μm2  that invP=0.012) higher than the 116 recorded in the ORT group. Moreover, the percentage of mice affected by lung metastases was also significantly (P<0.05) higher in the SC+ORT (89%) than in the ORT (30%) group. No other differences in lung metastases were recorded; thus, the number and size of macrofoci did not differ significantly between groups. No visible lung metastases were detected in either group .All primary tumours and metastatic foci from SW48 and HCT116 mice were verified to be human-origin tumour cells (HCT116 or SW48) by using the human-specific antibody MHC-1 class I (P=0.002) lower (1.74±0.25) number of apoptotic figures than the ORT (2.58±0.25) (P=0.002) than in the ORT group (10.9±1)  . Similar(10.9±1) . Interes(10.9±1) ; Table 3P<0.001) higher than the number of clusters (6.7±0.5/field) in the ORT group (4.1±0.4/field) (P<0.001) higher than in ORT tumours (1.2±0.2/field) (P=0.515). The number of single epithelial cells at the invasion front in SW48 SC+ORT tumours was also higher (2.8±0.4/field) than in the ORT tumours (2.4±0.4/field) (P=0.137).We also analyzed tumour budding and the number of single tumour epithelial cells at the primary tumour invasion front as a measure of the invasive capacity of the tumours. Thus, the number of tumour clusters at the invasion front  was sign4/field) . Similar2/field) . Primary2/field) , but thi4/field) , but, agsupplementary material Table S1). We confirmed this finding by ELISA. Thus, primary tumour tissue in the SC+ORT group displayed a significantly (P=0.004) higher level of expression of VEGF-A than in the ORT group. In contrast, VEGF-A expression was absent in both the SC−ORT and ORT groups in SW48 primary tumours , was increased as compared with ORT tumours  than in the ORT group . This activation was almost undetectable using IHC with an anti-p-AKT antibody in the SC+ORT and ORT groups in SW48-derived mice (P<0.01) lower levels of β1 integrin than in the ORT group , whereas, in SW48 tumours, no differences between groups were found . Moreover, no expression of the two analyzed epithelial-mesenchymal transition (EMT) markers, Snail1 and vimentin, was detected in either group. Similarly, the expression and subcellular localization of β-catenin did not change its pattern between groups (data not shown). Additional proteins that are associated with migration, invasion/and or metastases in CRC, such as serpine1, p-P38, p-MAPK, IL8, PTHLH and CD44v6, showed no significant differences in expression in primary tumour between groups (data not shown).in-vitro-established cell lines. In both CRC models, SC preconditioning enhanced, in an organ-specific manner, the number of microfoci (colonization) and/or its growth to macrofoci and visible metastases. In the HCT116 model, this procedure significantly increased the number and area of visible lymph node foci and enhanced colonization of the lung. It also increased colonization in the liver and enhanced the transition from micro- to macro-foci and to visible metastases. Interestingly, the animals with massive liver metastases died earlier than the other animals, a finding that is consistent with compromised liver function and death from liver metastases.We improved the metastatic yield of the HCT116 and SW48 CRC cell lines by using SC passage prior to orthotopic microinjection. In the case of HCT116, this procedure maintained the dissemination pattern at all clinically relevant sites . In the SW48 cell line, SC preconditioning generated metastases at lymph nodes, lung and peritoneum. This was in contrast to the direct orthotopic microinjection that generated a non-metastatic model. These results are consistent with our previous report of increased metastases in lymph nodes and bone marrow in a diffuse large B-cell lymphoma model after SC conditioning , suggestWe consider that SC preconditioning prior to orthotopic cell microinjection generates relevant models of metastases because they replicate the situation observed in individuals with CRC, who frequently die of metastases. In the case of the HCT116 model, this is especially relevant because they generate visible metastases that compromise mouse survival, as in humans . SW48 gein vivo passages: our model takes only 15–20 days to develop an SC tumour and about two or three additional months to obtain metastases in the subsequent orthotopic models.In addition, this procedure could be useful to study the mechanisms of metastases simultaneously in the several organs and the preclinical development of anti-metastatic drugs. It has the advantage of reducing experimental time as compared with the use of successive Metastatic dissemination was probably affected by a process that is common, and prior, to tumour cell dissemination towards all metastatic sites. This argument is in agreement with the higher number of surviving tumour cells (as assessed by the lower number of apoptotic figures observed) in HCT16 SC+ORT and SW48 SC+ORT primary tumours than in their respective ORT groups. It is also consistent with the higher number of keratin-positive single-tumour epithelial cells and higher number of tumour cells forming small clusters (tumour budding) at the invasion front in HCT116 SC+ORT primary tumours. The same trend towards increased invasion was also present in the SW48 SC+ORT tumour edge. However, it did not reach significance. Therefore, whereas in the HCT116 model both increased invasion and reduced tumour cell death can enhance metastatic dissemination, in the SW48 model the enhanced dissemination seems to depend mainly on reduced tumour cell death. In addition, the fact that tumour cell death rate in HCT116 tumours is around fourfold lower than in SW48 is in agreement with the lower metastatic capacity observed in SW48 mice than in HCT116 mice. This lower metastatic capacity of the SW48 model could be explained by the lower viability of these cells, as compared with HCT116 cells, when invading the tissue or disseminating through the bloodstream.Our findings agree with the enhanced metastatic dissemination and reduced survival associated with detection of single-tumour cells and tumour budding at the tumour invasive front in other CRC mouse models  and in CDetachment of single epithelial cells from the tumour mass has been associated with enhanced migration and invasion, and it affects both lymphatic and haematogenous dissemination . We asseVEGF-A was enhanced in the primary tumours of SC+ORT HCT116 mice as compared to ORT HCT116 mice . This is consistent with the overexpression of VEGF mRNA and protein associating with tumour progression and poor prognosis in CRC patients . It has in vitro and in vivo  and was cultured in DMEM  supplemented with 10% FBS , 50 units/ml penicillin and 50 mg/ml streptomycin .in vitro, and the other group (SC+ORT) received an orthotopic microinjection of cells disaggregated from SC tumours (HCT116-SC or SW48-SC) previously generated in a different set of mice. We used Swiss nude mice  for in vivo studies. The study was approved by the Animal Ethics Committee at Hospital de la Santa Creu i Sant Pau.In HCT116- and SW48-derived tumours, we compared primary tumour growth, invasion and metastasis development between two groups: one group (ORT) received a direct orthotopic microinjection into the cecum of HCT116 and SW48 cells grown 7 cells, in DMEM, in both flanks, in five mice. Tumours were measured every 2 days using a caliper. When tumours reached an approximate volume of 700 mm3, mice were sacrificed by cervical dislocation and SC tumours were excised. Necrotic areas were discarded. Tumour aliquots were taken for histological study as described  and 100 mg/ml DNase . The mix was pipetted 30 times using a 10 ml pipette, and incubated for 10 minutes at 37°C with shaking. It was then re-pipetted 30 times – using 10 ml, 3 ml and 1 ml pipettes – and re-incubated for 5 minutes at 37°C with shaking. This re-pipetting step was then repeated. The obtained HCT116-SC or SW48-SC single-cell suspension was filtered through a cell strainer and centrifuged at 1000 n=8–12) and subcutaneous+orthotopic  mice. We next injected 2×106 HCT116 cells or SW48 cells, previously grown in culture and resuspended in 50 μl of media, in the cecum of each mouse in the ORT group, following the method published by our group . Metastatic foci were considered macroscopic when their diameter exceeded 1 mm, meaning that the measured area in their tumour sections was higher than 785,000 μmroscopic . VisibleTo compare the invasive capacity of the ORT and SC+ORT primary tumours, we counted 40 fields (3–5 sections per tumour) at the tumour invasive front for each tumour group. After staining with anti-A1/A3 keratin, we recorded the number of keratin-positive single cells and keratin-positive tumour cell clusters, containing five or less cells (tumour budding), per 100× tumour field.H&E primary tumour sections of each group were used to quantify the number of apoptotic cells per field. Ten 400× fields per primary tumour were analyzed.The immunohistochemical analysis (IHC) was done on FFPE tissue. IHC stains were performed on a DAKO Autostainer automated Link48  using standard procedures.The metastatic foci were verified to be human-origin tumour cells (HCT116 or SW48) by using the human-specific antibody anti-MHC-1 . Samples were incubated with the corresponding primary antibodies using the following dilutions: integrin β1, β2, β3, β4, β5, α1, α2, α3, α4, α5, α6 and αv , p-AKT , ANGPT2 , p-MAPK , p-MAPK-38 , vimentin , PTHLH , VEGFA , CD44v6 , β-catenin , serpine1 , CXCR4 , anti-A1/A3 keratin , CD34 , E-cadherin , and then with mouse or rabbit secondary antibodies . We next incubated the sample with DAB substrate (DAKO) for 5 minutes and contrasted the staining using haematoxylin. IHC evaluation was performed by two independent observers, examining only areas with viable tumour. We analyzed the expression of each antibody in the bulk of the primary tumour, in the invasive front and in the metastatic foci in the ORT and SC+ORT groups. We quantified the percent of stained tumour cells in relation to the total cells and estimated the IHC staining intensity . We multiplied both values to obtain a final score of protein expression for each sample. This allowed statistical comparison between groups. Similarly, the number of single cells and cell clusters at the tumour invasive front was quantified by two independent observers.P<0.05.Mice that did not develop primary tumour or metastases in any organ were excluded from the analysis. The Fisher exact test was used to analyze statistically significant differences between primary tumour and metastatic take rates. The Mann-Whitney test was used to compare tumour size, number of apoptotic figures, single tumour cells, tumour clusters or metastatic foci between groups. Differences in survival between groups were evaluated using Kaplan-Meier curves and the Log-rank test. All quantitative values were expressed as mean ± s.e.m. and the statistical tests were performed using SPSS version 11.0 . Differences between groups were considered significant at


Local recurrence of glioblastomas is a major cause of patient mortality after definitive treatment. This review discusses the roles of the chemokine stromal cell-derived factor-1 and its receptor CXC chemokine receptor 4 (CXCR4) in affecting the sensitivity of glioblastomas to irradiation. Blocking these molecules prevents or delays tumour recurrence after irradiation by inhibiting the recruitment of CD11b+ monocytes/macrophages that participate in revascularising the tumour. We review the literature pertaining to the mechanism by which revascularisation occurs following tumour irradiation using experimental models. Areas of interest and debate in the literature include the process by which endothelial cells die after irradiation and the identity/origin of the cells that reconstitute the tumour blood vessels after injury. Understanding the processes that mediate tumour revascularisation will guide the improvement of clinical strategies for preventing recurrence of glioblastoma after irradiation. Despite advances in techniques for administering radiotherapy, local recurrence of glioblastomas (GBMs) typically leads to patient mortality. In this review, we focus on the chemokine stromal cell-derived factor-1/CXC chemokine ligand 12 (SDF-1/CXCL12) and its receptor CXCR4 in affecting the sensitivity of GBM to irradiation. We begin with an overview of the discovery and characterisation of these molecules, followed by an examination of their role in tumour metastasis and tissue injury. Finally, we discuss the data and controversies surrounding their role in revascularisation and tumour recurrence. Our understanding of the role of SDF-1 and CXCR4 in tumour recurrence post irradiation is largely gained from pre-clinical studies using mouse xenografts of human tumours. The pre-clinical data will be the focus of this review. Clinical studies in this area are limited, but serve as an exciting area for future investigation.The CXC chemokine family has multiple roles in leukocyte migration, immune response, angiogenesis, and tumour metastasis. CXC chemokines have four highly conserved cystine amino-acid residues, with a non-conserved amino acid separating the first two cystine residues. A second structural domain determines their function. The CXC chemokine family contains both angiogenesis-promoting and angiogenesis-inhibiting molecules. Stromal cell-derived factor-1 is regarded as an angiogenesis-promoting chemokine.2+ in response to human SDF-1. The mouse CXCR4 receptor was subsequently found by cloning candidate chemokine receptors and comparing the amino acid sequence with the human cDNA  was not examined. Chemotaxis of CXCR4-expressing murine muscle satellite cells towards SDF-1 has also been demonstrated, suggesting that migration of tissue-specific stem cells may be regulated by SDF-1 , a key proangiogenic molecule in circulating CD11b+ cells. We demonstrated that tumours cannot grow in an irradiated site (given 20 Gy) of an MMP-9 knockout (KO) mouse but can grow in a non-irradiated MMP-9 KO mouse. Tumour growth is restored following irradiation if the bone marrow in the MMP-9 KO mouse is replaced with wild-type bone marrow  or by circulating cells – including endothelial cells (ECs) or endothelial progenitor cells (EPCs), a process known as vasculogenesis (see the section ‘Restoration of tumour blood vessels after irradiation’). In pre-clinical models, several investigators have reported that radiation inhibits local angiogenesis, the generation of new vessels from surrounding vessels  following irradiation lead to accumulation of CXCR4-expressing monocytes/macrophages in the irradiated tumour. This is also suggested by data in the literature showing that the recruitment and retention of proangiogenic hematopoietic cells to sites of ischaemic tissue damage or to tumours is mediated by the interaction of the SDF-1 with CXCR4  or at TCD50 doses (doses that control 50% of the tumours) typical for transplanted tumours (40–100 Gy), and particularly in SCID mice in which all the stromal cells are highly radiosensitive . CD11b+ cells from an irradiated tumour share many of the same properties as ECs and have been shown to interact with newly forming tumour blood vessels  after a median follow-up period of more than 18 months. Although the majority of recurrences were located in the radiation field, in 17 patients GBM recurred outside the radiation field. As recurrences outside the radiation field were observed after a longer follow-up time, different mechanisms may be responsible for in-field and out-of-field recurrences . These strategies have been successfully used in pre-clinical studies. Of these, the use of plerixafor is currently the most promising for further investigations in human clinical trials as a proof of principle, as this drug is already approved for clinical use. Understanding the mechanism by which tumours revascularise after irradiation offers promising therapeutic strategies for treating GBMs.


Vascuologenesis is the de novo establishment of blood vessels and vascular networks from mesoderm-derived endothelial cell precursors (angioblasts). Recently a novel mechanism, by which some genetically deregulated and aggressive tumour cells generate "micro-vascular" channels without the participation of endothelial cells and independent of angiogenesis, has been proposed. This has been termed "vasculogenic mimicry" and has implications beyond angiogenesis and adds another layer of complexity to the current concept for the generation of tumour micro-circulation. We suggest this is common phenomenon in head and neck squamous cell carcinoma (HNSCC) cell lines and other aggressive tumour cell lines. We present experimental evidence of vasculogenic mimicry in HNSCC cell lines and compare them with other tumours and a positive control vascular cell line.The cell lines used were HUVEC, HN 2a, 2b (primary and metastatic tongue base squamous carcinoma cell line), HCT116 (colonic carcinoma cell line) and DU145 (prostate carcinoma cell line).Pilot experiments were undertaken to assess growth of a bank of tumour cell lines on (growth factor reduced) matrigel (Sigma) with standard media .A functional growth assay was performed by preparing the appropriate cell suspension in serum free medium plated onto either bare plastic or a well pre-coated with growth factor reduced type 4 collagen analogues.Phase contrast photomicrographs were taken at 4 hours and 24 hours. Image analysis was performed; particular features of interest were two dimensional area (surrogate of growth and migration), branch points and end point measurements (surrogate of intercellular complexity).There were observable differences in growth of the cells on laboratory plastic and collagen matrix. Tumour cells formed capillary like networks similar to HUVEC cells. Metastatic HNSCC cells lines were found to have vasculogenic properties similar to HUVEC cell lines when compared to cell lines from their corresponding primary tumour. The endothelial growth factor antibodies used did not inhibit or stimulate cell growth when compared to control but did discourage vascular mimicry. Other tumour cell lines also displayed this property.Tumour "vasculogenic mimicry" must still be regarded as a controversial issue whose existence is not proven. The clinical importance of this phenomenon however, is that it does explain the lack of complete efficacy of current anti-angiogenic treatments due to the added layer of complexity. It provides a feasible mechanism of early tumour vascular supply which can co-exist and incorporate with later angiogenic mechanisms. We suggest that "vasculogenic mimicry" maybe a common neoplastic phenomena which appears to also be dictated by the cells micro-environment. Its existence also suggests a further process that of the development of tumour mosaic vessels as the neo-vasculature integrates with the existing endothelial lined systems. Much attention has been focussed on the role of angiogenesis, i.e. the recruitment or co-option of new vessels into a tumour from pre-existing vessels such as capillaries and post-capillary venules. Currently, angiogensis is widely accepted as the mechanism by which tumours metastasize, however angiogenesis may not be the only mechanism by which tumours acquire a microcirculation. Maniotis et al. reported a novel mechanism by which some aggressive tumours acquire a blood supply and demonstrated the generation of micro-vascular channels by genetically deregulated and aggressive tumour cells without the participation of endothelial cells and independent of angiogenesis. This has been termed "vasculogenic mimicry" and has implications beyond angiogenesis and adds another layer of complexity to the current theoretical framework for the generation of tumour micro-circulation  at a seeding density of 2*10There were observable differences in growth of the cells on laboratory plastic and collagen matrix (P < 0.001), Figure . Tumour Metastatic cell lines (HN2B) were found to have vasculogenic properties similar to HUVEC cell lines when compared to cell lines from their corresponding primary tumour (HN2A). The clinical impact of this phenomenon will also be a function of tumour cell size enabling optimal stromal interaction as evinced by the growth of other tumour cell lines.The anti-endothelial antibody was not cytotoxic to any of the cell lines investigated. The antibody, however, did discourage vascular mimicry significantly P < 0.001). It may be hypothesized that the visible networks may not be tubes and not represent vascular channels but electron microscopy work suggests that they do [. It may Tumour "vasculogenic mimicry" Figure  must stiThe process of angiogenesis consists of multiple, sequential and independent steps with a vista of positive and negative regulators of angiogenesis being involved . HoweverThere is a consensus that the microcirculation of aggressive tumours is complex, and depending on the time of observation, could consist of mosaic vessels , co-opted vessels and/or angiogenic vessels. There is also strong evidence for the existence of an intratumoral, tumour-cell-lined, ECM-rich, patterned network that can provide an extravascular fluid pathway, now known as the fluid-conducting meshwork. The entire microcirculation in aggressive tumours is suggested to be made up of a combination of these elements, and it is the result of destructive tumour growth and remodelling. Alternatively, the PAS- and laminin-rich, fluid-conducting meshwork could be an early survival mechanism for nutrient exchange and the release of fluid pressure. This meshwork could eventually be replaced by endothelial cells from nearby angiogenic vessels or from the bone marrow. The fluid-conducting meshwork might provide a site for nutritional exchange for aggressive tumours, and might therefore prevent necrosis of the tumour. Alternatively, it might be analogous to an oedematous inflammatory response, in which increased blood pressure leads to the escape of fluid along connective tissue pathways in intra-tissue spaces. The complex geometry of the laminin-containing ECM covering that encases the spheroidal clusters of tumour cells could also form a suppressive shield against immune surveillance.Endothelial cells can generate tractional forces, appropriate matrices, cord-like tessellated structures because of a limited degree of cell-ECM attachment and a high degree of cell-cell attachment . The invEndothelial cell shape requirements in the context of growth and limited cell detachment typical of endothelial cell differentiation may be mediated by the mechanical properties of a continuous cytomatix, between the endothelial cell adhesion receptors and its nucleus and could determine the response of the cell to its changing architectural environment via a hard wired signalling mechanism. The extended ECM/cyto-matrix/nuclear matrix as a unit of structure/function is evidenced by comparable tubular structures formed in culture by highly invasive, interconnected primary and metastatic melanoma. These have similarities to cords or tessellations generated by a variety of other cell types including mammary epithelial cells on the appropriate substrate. It is assumed that more invasive cells generate cords similar to endothelium within the first few hours of culturing; patterned tubes completely acellular resemble the dimensions and patterning of networks in tumours ,8. HenceThe patterns of microcirculatory vessels have been shown to have a prognostic significance in melanomas with those with the presence of loops and networks doing worse ,9,10. HoIn criticism of our current study, the networks formed by the cultured cells do resemble microvascular channels but, fall short of being the compelling evidence of vascular mimicry published by Maniotis et al.  in theirclinical implications are, however, that there is still too little knowledge of how the many events are coordinated and integrated by the cancer cell, with conspiratorial help by the stromal component of the host. Before drug development can proceed with a legitimate chance of success, significant gaps in basic knowledge need to be filled. A tumour circulation not lined by endothelial cells would play an important physiological role in the maintenance and growth of aggressive tumours. This is observed in breast epithelial tumour cells which form disorganised masses which undergo phenotypic reversion to form acini resembling normal tissue. This reversion occurs simply by manipulating their surface receptors in a 3D assay. This continuous tumour extracellular matrix interaction and traction could contribute to tumour remodelling, its metastatic potential and vasculo-genetically patterned tumour derived abnormal vessels. The tubular structures formed in culture by highly invasive, interconnected primary and metastatic melanoma has similarities to cords or tessellation generated by a variety of other tumour cells on the appropriate substrate. Such vasculogenic triggers and their receptors could be the next target for refined pharmacological interventions in the treatment of metastasis, as important if not more than the recently trialled anti-angiogenic drugs. The important difference may be that they could be more specific and avoid the bystander cross receptor cardiac problems attendant with anti-angiogenic therapy.The The authors declare that they have no competing interests.All authors have contributed intellectually and to the writing of this manuscript. All authors read and approved the final manuscript.


Fumaric acid ester derivatives, or fumarates, have immunomodulating properties . For oveTo assess the response of fumarates in lupus erythematosus.Two case-reports of patients with lupus erythematosus who were treated with fumarates. Treatment was carried out with enteric-coated tablets containing 120 mg dimethylfumarate and 95 mg monoethylfumarate-salts. The dosage was gradually increased up to 6 tablets daily. Patients were followed up regularly with clinical evaluations, laboratory tests, and urine analysis.A 35-year old female patient presented with an erythematous indurated swelling of the nose that was present for 1 year. Histology of a skin biopsy was consistent with the diagnosis lupus timidus. Treatments included hydroxychloroquine 200 mg two times a day for four months and intralesional corticosteroid injections, but both treatments were without effect. Therefore, fumarates treatment was started. After 3 months the skin lesions resolved completely. At one year follow-up the clinical situation was stable with 4 tablets daily. Adverse events were nausea, diarrhea, and mild leukopenia and proteinuria, but these resolved gradually.A 24-year old female patient presented with erythematous scaly plaques, Raynaud's phenomenon, alopecia, and oral ulcera with positive antinuclear antibodies. She was diagnosed with systemic lupus erythematosus. Treatments with prednison, hydroxychloroquine, and cyclosporin were ineffective. Therefore, fumarates in combination with prednisone 10 mg was started, which resulted in improvement of the skin lesions. Because of a pregnancy wish, fumarates was stopped. Re-treatment with fumarates had an equally good effect within 3 months. Adverse events included tiredness, pruritus, abdominal cramps, and transient proteinuria.Fumarates seem beneficial in the treatment of lupus erythematosus not responding to conventional systemic treatments. The short-term side effect profile is similar to that in psoriasis patients.


Desmoplastic trichoepithelioma (DT) is a benign appendageal tumour predominately localized on the facial skin. The histological diagnosis can be difficult in some cases. Partial malignant transformation of a DT is a rarity and a complete transformation has never been described in literature.A DT of the upper lip was diagnosed histologically by a small biopsy 4 years previously. At presentation, the tumour had enlarged and had partly infiltrated the left side of the upper lip and subnasal region. Histological evaluation confirmed a microcystic adnexal carcinoma but without evidence of malignant transformation of the DT. It appeared that a too-small initial biopsy had led to the incorrect histological diagnosis of a benign tumour. Thus, it was necessary to perform a tumour resection and reconstruction using a two-flap technique including a rotation flap and an Abbé flap. Functional and aesthetic outcomes were good after 6 months. There were no recurrences during a 12-month follow-up.A facial DT should be resected completely. Patients should be attended for follow-ups, keeping in mind the difficulty of making a proper histological diagnosis from small biopsies or excisions and the consequences of ablative facial surgery. However, in particular cases, subtotal defects of the upper lip region are amenable to reconstruction without gross functional or aesthetic deficits. Desmoplastic trichoepitheliomas (DTs) amount to less than 1 % of all cutaneous tumours and belong to one entity of the various spectra of benign follicular differentiated appendageal neoplasms of the skin . In contA 56-year-old Caucasian man had been admitted to our clinic 4 years previously with a 1.2 × 0.5-cm nodular efflorescence of the left side of the upper lip. Biopsy and histological examination provided the diagnosis of a DT. The patient refused complete excision and follow-ups. Four years after initial diagnosis, the patient observed a rapid enlargement of the tumour. At presentation, a 2.8 × 2.4-cm, nodular, plaquelike tumour was found that had palpatorically infiltrated the upper lip and subnasal region Fig. . No recuPrevious case studies have reported the possibility of malignant transformation of a DT. In these cases, histopathology revealed a transformation zone between the DT and the malignant tumour mass as evidence of partial transformation . In the However, the reevaluation of specimens of the initial biopsy by pathologist conceded the case of misdiagnosis by comparing the growth pattern with that found in specimens obtained from the complete tumour excision. This was the most decisive point since infiltrative growth could only be found in deeper slices from the complete tumour specimen Fig. . The immHowever, a total resection of a MAC with clear surgical margins must be performed. Large defects following tumour resection in aesthetically and functionally sensitive areas, especially in the lip region, are still a challenge for reconstructive surgery, and they are discussed controversially in the literature. Reconstruction of a large defect of the upper lip can be performed by a variety of local flaps or by microvascular free-tissue transfer. In the present case, two local flaps for reconstruction of the upper lip and paranasal defects were performed during one operation. We were able to close the gross soft tissue defect with a rotation flap from the lower lip and cheek. A modified Abbé flap  was used


Aberrant expression of the progenitor marker Neuron-glia 2 (NG2/CSPG4) or melanoma proteoglycan on cancer cells and angiogenic vasculature is associated with an aggressive disease course in several malignancies including glioblastoma multiforme (GBM) and melanoma. Thus, we investigated the mechanism of NG2 mediated malignant progression and its potential as a therapeutic target in clinically relevant GBM and melanoma animal models. Xenografting NG2 overexpressing GBM cell lines resulted in increased growth rate, angiogenesis and vascular permeability compared to control, NG2 negative tumours. The effect of abrogating NG2 function was investigated after intracerebral delivery of lentivirally encoded shRNAs targeting NG2 in patient GBM xenografts as well as in established subcutaneous A375 melanoma tumours. NG2 knockdown reduced melanoma proliferation and increased apoptosis and necrosis. Targeting NG2 in two heterogeneous GBM xenografts significantly reduced tumour growth and oedema levels, angiogenesis and normalised vascular function. Vascular normalisation resulted in increased tumour invasion and decreased apoptosis and necrosis. We conclude that NG2 promotes tumour progression by multiple mechanisms and represents an amenable target for cancer molecular therapy. GBM is the most common and aggressive brain tumour in adults, where the median survival after diagnosis is only 14.6 months CSPG4 gene encoding the NG2 proteoglycan, is turned off upon terminal differentiation, but is aberrantly re-expressed by several tumour types 6-methylguanine methyltransferase (MGMT) DNA repair gene promoter . The association of high NG2 expression with an aggressive disease course may be mediated by its role in increasing cell proliferation, angiogenesis and treatment resistanceWe have previously shown that a subpopulation of GBM cells express Neuron-glia 2 (NG2/CSPG4), also known as melanoma proteoglycan (MPG), a cell surface chondroitin sulphate proteoglycan that is also expressed by numerous tissue specific progenitor cells during development. The Others have demonstrated high NG2 expression in 95% of uveal melanomas in vitro as spheroids in situ, such as diffuse invasion of the brain parenchyma, and cellular heterogeneity Several treatment approaches, such as anti-angiogenesis treatment and viral gene therapies CSPG4, were xenografted into nude rat brains and tumour progression compared to NG2 negative xenografts. NG2 expression enhanced the growth of the tumours compared to the NG2 negative controls (1). The NG2 positive tumours were larger, exhibited MRI detectable central necrosis and displayed similar characteristics to human GBMs in situ (2). Only obstructive cerebrospinal fluid (CSF) was apparent in the NG2 negative tumours (3), while peri-tumoural oedema was most evident in the rapidly growing NG2 positive tumours, as shown by T2 weighted MRI (4). Indeed, quantification of the solid tumour tissue and oedema revealed a significantly slower growth rate of the NG2 negative tumours, with mean volumes approximately 25% of those of NG2 positive tumours , in contrast to the NG2 negative tumours . Examination of the invasive edges in these tumours revealed microsatellite invasion of the brain parenchyma by positive NG2 tumour cells. U251-Wt tumours were uniformly NG2 negative , except for expression on oligodendrocyte progenitors in normal brain . To clarify whether the alterations in vascular morphology were also accompanied by functional changes, we estimated microvascular parameters such as permeability, fractional blood volume and elimination rate of contrast agent, as well as the extent of vasogenic oedema. There was a significant increase in tumour and oedema volumes in the U251-NG2 compared to the U251-wt tumours , and the decay phase of the curve is representative of the (K2)  heterogeneous contrast enhancement and necrosis on T1 weighted images after administration of gadodiamide (4). The heterogeneity of enhancement reflects the presence of both highly vascularised regions with leaky vessels and necrosis within the same tumour (4). Animals treated with control shRNAs exhibited similar MR features as the patient (2 and 2A5) as well as shift of midline structures and obstructive CSF due to the expansive tumour mass (2). The NG2 shRNA treated animals had smaller lesions with minimal MR signal intensity alterations in the tumour compared to NG2 expressing tumours (3 and 2A6). The mean tumour volume in the control shRNA treatment group was significantly larger than in the NG2 shRNA treated group, where additionally, not all animals developed MR detectable tumours . The invasive edge of the patient biopsy revealed diffuse invasion of tumour cells into the brain parenchyma (2). The histological features of control shRNA and NG2 shRNA treated P3 xenograft tumours were in accordance with the MR images (inserts in 1). The NG2 expressing tumours were highly cellular, contained pseudopalisading necrosis with surrounding pyknotic cells (1) and numerous small and large vessels (1). In contrast, the NG2 shRNA treated tumours frequently exhibited bilateral and microsatellite lesions that did not enhance contrast on T1 weighted images , where quantification of the vascular area fraction and vascular density proved that the NG2 shRNA treated tumours were significantly less vascularised . The number of NG2 positive tumour microvessels was also significantly reduced by the NG2 shRNAs compared to control shRNA treated tumours . The reduction in NG2 positive microvasculature was similar to the reduction in vWF positive vessels in the NG2 shRNA treated tumours. The differences in tumour vasculature were also reflected in the change in contrast enhancement ratio . Control shRNA treated tumours had a significantly higher contrast ratio between the tumour and brain tissue on T1-wighted MRI images after contrast agent injection  which predominantly expressed NG2 on the tumour vasculature. The tumours treated with control shRNAs were highly angiogenic, indicated by the presence of large, leaky and haemorrhagic vessels, with morphological features of “vascular lakes”, , and resp) in the tumour compared to control shRNAs, although this difference was not statistically significant . At the termination of the experiment A375 melanoma tumours treated with NG2 shRNAs had a mean volume of 1120.6±486.6 mm3 compared to 2690±807.3 mm3 for those treated with control shRNA and 3375.0±866.1 mm3 for those injected with PBS vehicle only. Immunohistochemical analyses confirmed high NG2 expression in the untreated parental and control shRNA treated tumours . Highly demarcated solid tumours , with ly tumours  and decrThe prerequisite for successful translation of experimental data to clinical practice is generation and validation in relevant model systems. The present study sought to validate the role of NG2 in malignant progression and its potential as a therapeutic target using the available animal models for preclinical therapies. We showed that NG2 expression affected several aspects of tumour progression and that targeting it in GBM and melanoma xenografts suppressed angiogenesis, and reduced tumour growth. NG2 overexpression induced highly aggressive tumours characterized by increased angiogenic and moderately invasive tumour phenotypes. Indeed, larger areas with signal alterations on T2 weighted and increased contrast enhancement on T1 weighed MR images showed that the NG2 positive tumours were more rapidly growing, with disrupted blood brain barrier integrity, and increased vascular volume fraction. We have previously used two pharmacokinetic models Increased vasogenic oedema was evident in the aggressive U251-NG2 tumours that also had a more invasive phenotype compared to the U251-Wt tumours. This finding was also confirmed in the P3 and P13 GBMs, where the control shRNA treated tumours exhibited higher levels of vasogenic oedema compared to NG2 shRNA treated tumours. Oedema contributes to morbidity and mortality, and is an independent predictor of outcome for GBM patients end-stage. These histological data and the MRI quantification showing reduced tumour volumes can be reconciled by the fact that MRI was undertaken 2–3 weeks prior to development of neurological symptoms. Indeed, three animals that did not have MRI detectable tumours were shown at post-mortem to harbour small, butterfly-like, non-angiogenic tumours. Thus, at end-stage tumours from both treatment groups were in an exponential growth phase characterised by rapid proliferation rates, hence the lack of statistical significance on histological analyses. Furthermore, tumour volume quantification by T1 weighted MRI alone may not always be accurate to define the tumour mass in the absence of blood brain barrier disruption and leakage of contrast agentTherapeutic strategies combining tumour and vascular targets are hypothesized to be more successful than targeting either compartment alone. As a membrane glycoprotein expressed on tumour cells and pericytes, NG2 is an attractive candidate for targeting both the tumour and angiogenic compartments within the tumour. P3 GBM xenografts expressed NG2 on both tumour cells and their angiogenic vessels and had a moderately invasive phenotype. Disruption of NG2 function in these xenografts significantly reduced tumour growth and angiogenesis by approximately 70% and 50%, respectively. However, there was no significant difference in cell proliferation and apoptosis measured at in vivo, where the ensuing hyperemia results in leakage of plasma proteins, and sustained recruitment of leukocytes and macrophages. In contrast to the melanoma tumours, NG2 shRNA treatment of both P3 and P13 GBM xenografts did not significantly reduce tumour proliferation. We postulate that this maybe due to normalisation of the angiogenic vasculature as indicated by diminished leakage of the contrast agent as detected by T1 weighted MR images. DCE-MRI with pharmacokinetic modelling revealed increased tracer efflux from the interstitium in the P13 tumours treated with NG2 shRNAs. The finding of reduced oedema, decreased fractional plasma volume fraction and area fraction of vWF positive vessels in both patient GBMs further supports this finding. As previously described, the Ktrans parameter is influenced by both tumour vascular perfusion and permeability. Vascular normalisation would increase tissue perfusion, leading to an increased Ktrans parameter as we indeed observed. Moreover, the relatively small change in the plasma volume fraction maybe due to the contribution of blood flow that masks the true plasma volume fraction in the NG2 shRNA treated tumours. Vessel normalisation also improves oxygenation. Taken together, these factors would contribute to increased tumour cell proliferation, reduced hypoxia and necrosis. Indeed, P13 tumours treated with NG2 shRNAs had significantly reduced apoptotic cells indicated by TUNEL labelling. In contrast to the GBM xenografts, NG2 shRNA treatment may have drastically reduced neovascularisation in the melanoma tumours, thereby cutting off oxygenation and nutrient supply resulting in apoptosis and arrested cell cycle progression. Although NG2 shRNAs did not prolong survival, its drastic effects on vascular normalisation might make targeting NG2 expressing cells useful as neo-adjuvant treatment in combination therapy to permit more efficient delivery of systemically administered chemotherapy drugs to the brain and to sensitise the tumours to radiotherapy (RT). Vascular normalisation would sensitise to RT by re-oxygenating the tumour. Hypoxia is known to promote radioresistance by reducing cell cycle progression, thus increased blood flow would promote tumour cell redistribution in the cell cycle and thus sensitise them to IR. Increased blood flow might also increase the bio-distribution of systemically administered chemotherapy to the tumour. Reduced oedema would also reduce tumour interstitial pressure that would prevent collapse of blood vessels and permit appropriate perfusion of therapeutic agents from the blood to the tumouŕs extracellular space. Indeed, anti-angiogenic compounds have been employed to normalize the chaotic tumour vasculature resulting in more efficient delivery of therapeutic substances to the tumour bed Disruption of NG2 function in the melanoma model resulted in reduced proliferation, increased apoptosis and immune infiltration by macrophages, plasma cells and histiocyte mediated granulomatous response (data not shown). Dying cells trigger inflammatory responses 7/ml, NG2 was evidently expressed on a proportion of the angiogenic vessels post shRNA transduction indicating incomplete target inhibition. Multiple injections of the shRNA constructs, even higher viral loads and reduced implanted tumour burden might enhance NG2′s inhibitory effects on tumour growth, potentiating greater survival differences. The present study utilized this method of delivery as a proof of principle approach to demonstrate that abrogation of NG2 function by gene silencing diminishes tumour growth and angiogenesis. Previous studies indicated that targeting NG2 positive tumour cells using monoclonal antibodies (mAb) conjugated to diphtheria toxin may have therapeutic potential Although gene therapy trials using lentiviral vectors at present cannot be conducted in patients due to safety and bio-distribution concerns. Delivery is a major challenge for viral gene therapy strategies to the brain. Incomplete shutdown of NG2/CSPG4 mRNA translation in the total tumour mass by the lentivirally delivered shRNA constructs, inappropriate timing of the treatment with respect to tumour growth kinetics may have contributed to the lack of survival advantage after treatment. Despite high viral multiplicity of infectious units (MOI) greater than 5×10in vitro as previously described The human glioblastoma (GBM) cell line U251N  was transfected with the NG2 cDNA by calcium phosphate transfection using standard protocols.The cells were propagated as multicellular spheroids in medium supplemented with geneticin to maintain transgene expression as previously describedin vivo passaged through 21 and 22 generations of nude rats (respectively) were implanted into 15 and 16 (respectively) nude rat brains as previously described 5A375 melanoma cells.The National Animal Research Authority in Norway approved all animal experiments. Athymic nude rats (Han: rnu/rnu Rowett) and NOD-SCID mice were used in the GBM and melanoma studies, respectively. The animals were bred in an isolation facility at 25°C (55% relative humidity) in a specific pathogen free environment and animal husbandry protocols were maintained as previously described5′-GGGCUGUGUUGAAGAGUUU-3′. Target sequence of the scrambled control shRNA is 5′-GUAGAUCAAUUGGGUACACUU-3′. Lentiviral particles were produced by triple transfection of the lentiviral expression plasmids encoding shRNA, together with vsv-g encoding envelope plasmid (pMD2.G) and a gag-pol encoding packaging plasmid (psPAX.2) into HEK 293FT cells. Envelope and packaging plasmids were kindly provided by Tronolab and protocols for virus production and harvest is largely adapted from their website http://tronolab.epfl.ch. Briefly, lentiviral particles were harvested 48 h and 72 h post-transfection, sterile filtered, and concentrated for in vivo experiments by polyethylene glycol (PEG) 6000 precipitation Lentiviral vectors targeting NG2 were produced by subcloning the selected shRNA sequences into the pRNAt-U6.1/Lentiviral vector from GenScript . The NG2 shRNA targets the following site: Monolayer cultures of primary GBM biopsy material p3 and p13 were cultivated in serum-free neurobasal medium (Invitrogen), supplemented with B-27 supplement (Invitrogen) and 20 ng/ml FGF and 20 ng/ml EGF. Viral infection was performed by centrifugation of cells in the presence of viral particles at 1200 g for 90minutes at 31°C. In vitro NG2 knockdown was validated by Western Blotting, whilst in vivo knockdown was validated by immunostaining for NG2 on tissue sections of the treated tumours. Samples of infected monolayers harvested 96 h post-infection.cspg4 mRNA sequence in both P3 and P13 cohorts. After infusion, the needle was left in place for 5 min and thereafter slowly retracted and the skinfolds closed with polyamide surgical thread.Two-three weeks after tumour implantation, the nude rats were MRI scanned to confirm tumour engraftment. The rats were anesthetized and a craniotomy performed as previously described V) were calculated using the formula (V) = (a×b×c)/2, where a, b and c are the long axis, short axis and the depth, respectively, that was derived for an ellipsoid (πd3/6) as previously describedFourteen melanoma bearing mice received intra-tumoural injections of control or NG2 shRNAs, while 7 received PBS vehicle 7 days after tumour implantation. Following surgery, the animals were monitored until recovery in an incubator at 35°C. Melanoma tumour growth was determined by measuring tumour nodules with a caliper, and tumour volumes  and U251-wt (n = 4) tumours were imaged with a small animal horizontal bore 2.35T Biospec  equipped with a specially designed rat head coil. T2 weighted (T2 w) spin echo (SE) images and post-contrast T1 weighted (T1 w) SE images were acquired for vasogenic oedema and solid tumour volume measurements respectively (repetition time (TR)/echo time (TE) were 500/113 ms and 524/13 ms, respectively). Both sequences were acquired using 4 NEX (number of acquisitions), matrix of 256×256 and 1 mm slice thickness. To assess microvascular status, we performed dynamic contrast enhanced MRI (DCE-MRI) using a dynamic T1 weighted SE sequence with 100 repetitions and 12 s temporal resolution in 4 axial slices . Gadolinium-based contrast agent  was administered in the femoral vein after baseline images were acquired, and an additional dose of 0.3 mmol/kg the contrast agent was given 4 min prior to the post-contrast T1 weighted sequence. Fifteen animals bearing P3 GBMs were scanned at 2 months, while 13 animals bearing P13 tumours were scanned two weeks after tumour implantation and 2 weeks post treatment using a 7T Pharmascan MR scanner . Pre- and post-contrast T1 weighted RARE (Rapid Acquisition with Relaxation Enhancement) sequence , and a T2 weighted RARE sequence  were performed. DCE-MRI was performed on animals bearing human GBM from Patient 13 to assess functional changes in the microvascular status after treatment using a FLASH-sequence (Fast low angle shot) with a flip angle of 25° (TR/TE = 16.3/2.8 ms) and 550 repetitions with a temporal resolution of 1.5 s . A bolus injection of gadodiamide (0.1 mmol/kg) was administered after acquisition of 30 baseline images. All sequences were obtained with identical geometric imaging parameters. The tail veins were cannulated to ensure secure injection of the contrast agent.tumour- SIhealthy brain)//2), where the SI in the healthy brain tissue was measured in the contralateral hemisphere. The DCE-MRI data were analysed by applying the pharmacokinetic model proposed by Su et ala priori based on increase in signal intensity of the tumour after contrast agent injection. When applying Su's model, the signal intensity time curves were extracted and curves from regions-of-interest (ROI) were converted to tissue concentration based on the assumption that the increase in T1 relaxation rate is proportional to the concentration of the tracer. The microvascular parameters D0 , D1  and K2  were estimated. For the Tofts model, nordicICE was used to estimate pixel-by-pixel the transfer constant, Ktrans  and transport across the vascular endothelium (by permeability), with the dominant factor depending on whether the tracer depletion is flow or permeability limited), ve , vp (plasma volume fraction) and the Kep . In a previous study Volumes of solid tumour tissue and oedema were estimated using image segmentation based on signal intensity threshold in T1 weighted post contrast and T2 weighted images, respectively, using nordicICE version 2.3.7 . The contrast ratio between solid tumour and healthy brain tissue in post-contrast T1 weighted images was estimated using the following equation: (SIMaximum intensity projection images from T1w MR images were generated using multiplanar reconstruction and the maximum intensity projection (MIP) tool in nordicICE. Manual reorientation in multiplanar reconstructed images and MIP was recorded in real time using CamStudio™ Open Source Free Streeming Video Software.Formalin fixed paraffin embedded sections from each specimen were stained with Harris Haematoxylin and Eosin  according to standard procedures and examined with a Nikon Eclipse (E600) light microscope. Images were captured with a Nikon DXM1200 digital camera . Indirect immunohistochemistry was performed using the EnVisionSystem, horse-radish peroxidase (HRP) and 3′3′-diaminobenzidine (DAB)  method, as described previouslyThe rat brain sections showing maximal tumour were imaged using a Nikon TE2000 fluorescent microscope (Nikon). To cover the whole brain, 9–20 images per section were acquired using a Plan apo 2x 0.1 NA objective. Images were merged together using Adobe Photoshop CS4 extended (version 11.0.1) through the automated photomerge function.The two-tailed Student's t-test was used when comparing two categories and one way- or two way-analysis of variance (ANOVA) for comparisons of more than 2 groups or variables. Mann Whitney-U test was used to analyse non-parametric data and the Kaplan Meier survival curves were analysed using the Log-rank (Mantel-Cox) test. A probability level of ≤0.05 was considered significant and all statistical analyses were performed in Graphpad Prism 5.0 software.Figure S1Correlation of MRI and Histology. Panel 1: Large, expansive H&E stained control shRNA treated tumour with pseudopalisading necrosis and shift of the midline structures. Panel 2: Small NG2 shRNA treated tumour disseminating to the contralateral hemisphere. Inserts in both panels show the area with signal change in T1 weighted images superimposed onto T2 weighted MR images.(TIF)Click here for additional data file.Video S1Video recorded in real time using multiplanar reconstruction and MIP from T1 weighted MRI post contrast images of control shRNA tumours demonstrating differences in tumour volumes and contrast enhancement.(AVI)Click here for additional data file.Video S2Video recorded in real time using multiplanar reconstruction and MIP from T1 weighted MRI post contrast images of NG2 shRNA treated tumours demonstrating differences in tumour volumes and contrast enhancement.(AVI)Click here for additional data file.


There was an error in the funding statement that was introduced during the production process. The correct funding statement is: This work is supported by the National Science Council, Taiwan, Republic of China, under grant numbers 95-2314-B-320-009-MY3, 98-2320-B-0.17-001-MY3 and 99-2320-B-017-002 -MY3, by the Ministry of Economic Affairs, Taiwan, Republic of China, under grant number 98-EC-17-A-19-S2-0111 and by Tzu-Chi University under grant numbers TCIRP 95002-02, TCIRP 98001-01, TCRPP 99020 and TCRPP 100003. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


An eight-year-old boy was admitted for evaluation of upper abdominal pain and fever. One year before the admission, he had an episode of subacute intestinal obstruction, which was managed conservatively. During current admission; on physical examination, we noticed a vague upper abdominal nontender distention but no definite palpable abdominal lump. Hemogram was within normal limit except leucocytosis. Serum amylase was markedly raised, and serum lipase was marginally increased. X-ray abdomen erect was suggestive of ground glass appearance with scanty amount of air in abdomen and without any pathological air-fluid level. Ultrasound abdomen showed fluid collection in between the stomach and pancreas. Computer tomography with the administration of oral contrast medium revealed a cavity in hepatogastric fossa with an air-fluid level. The gastrointestinal contrast material had not entered the cavity .What is the diagnosis?The computer tomography findings were consistent with a subhepatic abscess cavity with an air-fluid level. There was no visible communication with the bowel. Patient had managed by percutaneous drainage of pus cavity under intravenous antibiotic cover and he responded well to the treatment.Despite advances in surgical technique and medical treatment, intra-abdominal abscesses remain a common diagnostic problem. An air-fluid level may indicate the presence of a fistulous communication to the gastrointestinal tract, but its absence does not necessarily mean there is no communication.Gas may appear as bubbles in deep, if the medium is thick. In cases of thin material, gas may rise to the surface, forming either superficial bubbles or air-fluid level. Distribution of gas in an intra-abdominal abscess is associated with drainability. Abscesses with superficial gas  have a greater chance of being drained successfully than do abscesses with deep trapped gas.In abscesses with classic clinical features and focal abdominal tenderness, ultrasound is a rapid and sensitive diagnostic technique. Enteric communication with abscesses has been shown by barium studies and more recently by CT. The degrMortality may reach 100% without proper diagnosis and abscess drainage, despite antibiotic therapy.


Wellbore instability is one of the major problems that hamper the drilling speed in Halfaya Oilfield. Comprehensive analysis of geological and engineering data indicates that Halfaya Oilfield features fractured shale in the Nahr Umr Formation. Complex accidents such as wellbore collapse and sticking emerged frequently in this formation. Tests and theoretical analysis revealed that wellbore instability in the Halfaya Oilfield was influenced by chemical effect of fractured shale and the formation water with high ionic concentration. The influence of three types of drilling fluids on the rock mechanical properties of Nahr Umr Shale is tested, and time-dependent collapse pressure is calculated. Finally, we put forward engineering countermeasures for safety drilling in Halfaya Oilfield and point out that increasing the ionic concentration and improving the sealing capacity of the drilling fluid are the way to keep the wellbore stable. The Nahr Umr Shale Formation is found throughout the southern part of the Arabian Gulf and forms the cap rock to many major reservoirs in the region . Major wThe Halfaya Oilfield is in the south of Missan province in Iraq, which is 400 km south east of Baghdad, the capital of Iraq. Three horizontal wells in the Nahr Umr Formation of Halfaya Oilfield had been drilled. But, two wells of the three horizontal wells have sidetracking due to sticking, only one horizontal well is drilled successfully, so it illustrates the big effects of wellbore instability problems on the directional drilling in this oilfield.The Halfaya Oilfield is located on the Arabian shelf, which is adjacent to the Zagros tectonic zone. The influence of Zagros tectonic movement is the extrusion to the Arabian shelf by the European plate (NNE-SSW). The propagation of the stress wave leads to a series of anticlines in the Arab shell. This extrusion stopped in Middle Miocene. The geological structure is a low dip anticline, in which the long axis is nearly perpendicular to the Zagros extrusion stress field –4. The sThere is no large fault which could be recognized by seismic data. The anticline structure is also very smooth. The results show that the extrusion stress by the Zagros tectonic movement is not very strong, and the extrusion stress does not produce strong in situ deformation and destruction.The lithologic characters in the Halfaya Oilfield from the top to the bottom is, respectively, the Tertiary Upper Fars Group, mainly sandy mudstone, about 1300 m thick; the Lower Fars Group, mainly anhydrite, salt rock, and shale deposit, about 500 m thick, being the regional cap rock; the Tertiary Kirkuk Group which is mainly sandstone and mudstone, about 300 m; from the Tertiary Jaddala group to the Nahr Umr group, mainly carbonatite and interlayers of thin marl, sandstone and shale. Three horizontal wells, N001H, N006H, and N002H, in the Nahr Umr Formation of Halfaya Oilfield had been drilled. The well distributions are both located in the structural long axis direction. The complicated drilling problems of these three wells are as follows.When the first horizontal well (N001ST well) encountered the Nahr Umr layer, there were two sidetracking operations. The first sidetracking happened at 3941.26 m, the SLB screw stuck in the highly deviated interval and the directional tool dropped in the well. The fishing failed, which led to sidetracking. The second sidetracking happened at 4091.21 m, the Nahr Umr Shale collapsed and this led to sticking at 4087 m the treatment measures was ineffective and a sidetracking operation happened.The second horizontal well (N006ST well) used the organic salt drilling fluid which has a strong inhibition. When drilling 3964 m, the Nahr Umr Shale collapsed and the treatment measure for the sticking failed, so sidetracking happened at 3800 m using the vertical well completion.The third horizontal well N002H used saturated salt water drilling, but there were many sticks between 3660 m and 3895 m in the Nahr Umr Formation, and there were cavings at the shaking screen.The two wells of the three horizontal wells have sidetracking due to sticking, only one horizontal well was drilled successfully, so it illustrates the big effects of wellbore instability problems in the oilfield. The wellbore instability has serious impact for oilfield drilling, and it restricts the exploration and development in the oil field. As the development wells are generally directional wells or horizontal wells, and the wellbore instability risk is great, wellbore stability analysis is needed in the Halfaya Oilfield. The main reason for wellbore instability in hard brittle shale with lots of fractures is as follows –11. If tIn order to solve the wellbore instability of the fractured and hard-brittle shale in Nahr Umr Formation, the drilling fluid property and engineering countermeasures should be taken into account. We analyze the wellbore instability reasons combined with the drilling engineering measures of three horizontal wells in the Nahr Umr Formation.When using rational engineering measures, the drilling fluid property decides the wellbore stability during drilling to a large extent. (1) Based on the mud rheological parameters, for the formation with good completeness, the rheological parameters of these three wells are similar and could meet the engineering requirement. But, for the fractured shale formation, the rheological parameters of these three wells are different. Compared to the other two wells, the drilling fluid of N001H well has a low viscosity, which is bad for carrying the cuttings and cavings. In addition, the drilling fluid with low viscosity will easily flow into the formation under the pressure difference. Therefore, the rheological parameters of drilling fluid of N002H well benefit wellbore stability. Usually increasing the drilling fluid viscosity is of benefit for fracture formation.(2) Based on the drilling fluid filter loss, the filter losses of these three wells are similar. Because the filter loss is measured by the experimental instrument in the laboratory, the results cannot reflect the real formation situation and it is only a reference index.(3) Based on the drilling fluid ionic concentration, although there are not ionic concentration parameters of N001H well drilling fluid in the daily drilling report, according to the drilling fluid description provided by the drilling fluid service provider, the drilling fluid ionic concentrations of this well could indicate that the ionic concentration of KCL Polymer drilling fluid used in the N001H well is between the concentrations of the N002H well and N006H well; the ionic concentration of the N002H well is the highest the ionic concentration of the N006H well is the lowest. When the hole is opened, the ionic concentration difference of the drilling fluid and formation water is the main driving force that drives the free water in the drilling fluid into the formation. Commonly, the high ionic concentration of drilling fluid is of benefit to prevent the free water in the drilling fluid from flowing into the formation. If the free water in the drilling fluid flows into the formation, the formation will be hydrated, and the formation strength will be decreased, so as to lead to wellbore periodic collapsing. According to the formation character, the Nahr Umr Shale is abundant in microfractures; the formation is broken, and the drilling fluid can easily flow into the micro fracture plane, which leads to the change of formation strength. In order to prevent wellbore instability, the drilling fluid property should be improved. The influence of drilling fluid on the wellbore stability is analyzed from the mineral composition, the drilling fluid consistency, and the influence of the drilling fluid on formation strength.Tables According to research experience, if the drilling fluid inhibition is good enough, a formation like the Nahr Umr Shale is impossible to hydrate without expansion collapsing. Therefore, we evaluate the rejection capacity of three drilling fluid systems which are used in the Halfaya Oilfield. The three drilling fluids are organic salt drilling fluid, Gel-polymer drilling fluid, and KCl-polymer drilling fluid.3; then we measured the cuttings recovery  and swelling ratio of these three drilling fluids. The results are shown in The densities of these three types of drilling fluid are all 1.33 g/cmIn order to analyze the influence of the drilling fluid on the wellbore stability of the Nahr Umr Shale, experimental studies were carried on the influence of drilling fluid on the rock mechanical property. We tested the shale strength of Nahr Umr Shale after immersing it in different kinds of drilling fluids. Under the drive force of the ionic concentration difference, the free water in the drilling fluid which flows into the formation would decrease the rock strength, which is the main reason for the collapse in Nahr Umr Shale; in addition, as the formation is extremely hard and brittle and the fractures are rich internally, if the drilling fluid sealing capacity is not good enough, the drilling fluid and filtrate would flow into the rock along the microfracture under the difference of the drilling fluid column pressure and pore pressure, so as to weaken the formation strength and lead to wellbore collapse. So, the increasing of the ionic concentration of the drilling fluid and enhancing the drilling fluid sealing capacity is the key to the wellbore stability of the Nahr Umr Shale.According to mechanical concepts, the main reason for borehole collapse is caused by shear failure for the reason that stresses loaded on rock around the borehole exceed the rock strength, as a result of lower mud column pressure. Now, brittle formation collapse will generate and the borehole will enlarge; for plastic formation, plastic deformation be will generated and borehole shrinkage will be encountered.θ = π/2 or 3π/2 [σn as principal stress σ1 and σ3:Generally, borehole collapse takes place in the minimum horizontal stress direction,  or 3π/2 ; the bor or 3π/2 –20 is ascient FS  is as foriterion :(5)M/(1 − ν)) − ϕ; under mud penetrating borehole face, the borehole collapsing pressure model isBased on different borehole stress conditions, borehole collapsing pressure expresses as a different form. When the bearing condition is For the hard-brittle shale of Nahr Umr Formation, according to the study results and experimental results, the influence of the drilling fluid immersion on the mechanical property mainly reflects in the decrease of the compressive strength as the immersing time increase. Depending only on the drilling fluid density cannot solve the wellbore stability of the shale formation which is full of fractures , 23. If Commonly, the larger the inclination, the more possible the wellbore instability. But for the laminar fracture formation, decreasing the angle of the wellbore axial line with the bedding normal direction is of benefit for the wellbore stability.The influences of the swabbing pressure and surge pressure should be taken into consideration when evaluating wellbore stability; the simplified bottom hole assembly (BHA) could prevent large swabbing pressure and surge pressure and then prevent sticking.The hydraulic jetting is not suitable, because the high pressure hydraulic jetting would produce water wedge effect in the progress of the drilling seepage. The big diameter jet or no-jet are welcomed.Avoiding the intense change of the dogleg or the well track so as to prevent big drill string acting force to the wellbore wall.Optimizing the hydraulic parameters so as to ensure the cuttings could be carried out of the wellbore timely. For some situations, wellbore collapse can not be prevented, so carrying out the cuttings in a timely way could decrease the downhole complicated time. Increasing the drilling rate could decrease the exposed time of the shale formation, which is useful for the wellbore stability.The formation water has an extremely high ionic concentration, so keep a high ionic concentration for the drilling fluid to balance it.In order to prevent the wellbore instability of Nahr Umr Shale, we come up with the following drilling technology countermeasures and suggestions.Under the function of the ionic concentration difference, the free water in the drilling fluid which flows into the formation will decrease the rock compressive strength, which is the main reason for the collapse in Nahr Umr Shale; in addition, as the formation is extremely hard and brittle and the fractures are internally rich, if the drilling fluid sealing capacity is not good enough, the drilling fluid and filtrate will flow into the rock along the micro fracture surface under the difference of the drilling fluid column pressure and pore pressure, so as to weaken the formation strength and lead to wellbore collapse. So, increasing the ionic concentration of the drilling fluid to enhance the drilling fluid sealing capacity is the key point to the wellbore stability of the Nahr Umr Shale Formation.The collapse pressure will increase as the formation strength decreases after drilling; the increasing speed decreases gradually. The increasing rate of Gel-polymer drilling fluid is the lowest; in a certain drilling fluid density it can keep the wellbore stable for the longest time. The increase of the mud density could only keep the wellbore stability for a limited time. Improving the property of the drilling fluid is the basis for keeping the wellbore stable.