SCLC cells are of neuroendocrine origin and express high levels of somatostatin receptors; therefore, future treatment might involve targeting tumours with radiolabelled somatostatin analogues. This therapy induces abundant necrotic patches that contain exposed keratins; thus, keratin 8, which is one of the most abundant cytoskeletal proteins may represent an interesting secondary target for SCLC. This study aimed to investigate the effects of177Lu-DOTA-Tyr3-octreotate administered 24 h apart. Mice xenotransplanted with NCI-H69 were sacrificed 1, 5, 12, 20 and 150 days post-injection or when the tumour had regrown to its original size. GOT1-xenotransplanted mice were sacrificed 3 days post-injection. Immunohistochemistry was performed to evaluate TS1 staining in tumours and in seven human biopsies of primary SCLC from pulmonary bronchi. Central cell density and nucleus size were determined in NCI-H69 sections.NCI-H69- and GOT1-xenotransplanted mice were treated with three doses of 30 MBq177Lu-DOTA-Tyr3-octerotate treatment, the SCLC xenograft response was extensive. Twenty days after treatment, one of three analysed tumours displayed complete remission. The other two tumours showed 1/4 the cell density of untreated controls and cell nuclei were about three times larger than those of untreated controls. At 150 days after treatment, one of four mice exhibited complete remission. Treated tumours displayed increased TS1 antibody accumulation and high TS1 binding in necrotic patches. All seven human SCLC biopsies displayed necrotic areas with TS1 staining.Twelve days after177Lu-DOTA-Tyr3-octreotate had pronounced effects on tumour cell density and cell nuclei, which indicated mitotic catastrophe. Despite these anti-tumour effects, two of three SCLC tumours recurred. Further studies should investigate the nature of tumour cell survival and develop more effective treatments. High TS1 accumulation in tumour sections in vitro after177Lu-DOTA-Tyr3-octerotate treatment indicated that TS1 might represent a promising secondary therapeutic strategy.Radiation treatment with three injections of 30 MBq Dosimetric studies revealed that the therapeutic radionuclide,177Lu, had physical properties beneficial for therapy [177Lu emits medium-energy electrons, it is suitable for treating a wide range of tumour sizes. Its long physical half-life of 6.7 days and its higher retention in tumours compared to normal tissues provides an optimal ratio of tumour to normal tissue dose absorption. In a study by Schmitt et al., nude mice that bore tumours of the human SCLC cell line, NCI-H69, were used as a preclinical model. The results demonstrated that single doses of 45-120 MBq of177Lu-DOTA-Tyr3-octreotate caused extensive tumour regression during the first 1-3 weeks after treatment [177Lu-DOTA-Tyr3-octreotate given 48 h apart caused more extensive tumour regression. The tumours continued to decline over the entire study period (34 days) [177Lu-DOTA-Tyr3-octreotate treatment caused an up-regulation in somatostatin receptor mRNA expression. That result supported the idea that fractionated doses might induce receptor expression and, consequently, increase the uptake of177Lu-DOTA-Tyr3-octreotate [Small-cell lung carcinoma (SCLC) comprises about 15-20% of all diagnosed lung cancers. The prognosis of this disease is often poor; distant metastases are typically observed at the time of diagnosis. New improved treatment modalities are urgently needed and have been intensively discussed . SCLC is therapy ,26. Becareatment . In the 34 days) . However34 days) . Therefotreotate ,29. Howe131I-radiolabelled monoclonal antibody, thrombospondin-1 (TS1) [177Lu-DOTA-Tyr3-octreotate might further increase the cytoplasmic release of K8 and thereby increase the accumulation of radiolabelled TS1 antibodies in SCLC tumours.We hypothesised that somatostatin therapy combined with an anti-keratin antibody might comprise an effective SCLC treatment. The-1 (TS1) , which b-1 (TS1) . K8 is a-1 (TS1) . It has -1 (TS1) . In addi-1 (TS1) . The ant-1 (TS1) . Because-1 (TS1) , radiola177Lu-DOTA-Tyr3-octreotate treatment. We analysed changes in tumour cell density, nuclear size, and binding of TS1, Ki67, and Bcl-2 at different time points. We also confirmed TS1 binding in a177Lu-DOTA-Tyr3-octreotate-treated xenografts of the midgut carcinoid cell line GOT1 and in tumour biopsies from seven patients with small-cell carcinoma in pulmonary bronchi.The main purpose of this study was to investigate the binding of TS1 in small-cell lung cancer xenografts after7 cells of the human SCLC cell line, NCI-H69  or the midgut carcinoid cell line GOT1 [ad libitum. Tumour growth, animal condition, and body weight were determined at regular intervals. The animals were killed when they lost > 10% of their original body weight or when the tumour reached a diameter > 20 mm.Four-week-old male BALB/c mice  underwent subcutaneous implantations in the neck with 2 × 10ine GOT1 . TumoursThis study included biopsies from seven cases of human small-cell carcinoma in pulmonary bronchi that had not been treated with radiolabelled somatostatin analogues. Biopsies were fixed in formalin, embedded in paraffin, and cut into 4-μm-thick sections. Sections from all seven tumours were stained immunohistochemically with TS1 and sections from two tumours were analysed with anti-SSTR2. The tissue material was decoded before processing to remove links that could identify patients.3-octreotate  was dissolved in a 0.05 M HCL solution that contained 126 mg/ml sodium ascorbate and 25 mg/ml gentisic acid  [DOTA-Tyric acid) ,18.177LuCl3 with 740 MBq/μg maximal specific activity was obtained from Perkin Elmer .177LuCl3 was diluted to 4.2-5.3 MBq/μl and incubated with the DOTA-Tyr3-octreotate solution for 30 min at 80°C. This resulted in DOTA-Tyr3-octreotate with a specific activity of 30 MBq/μg. With instant thin layer chromatography  performed as described previously [177Lu was bound to peptide.eviously , we dete177Lu-DOTA-Tyr3-octreotate in 0.1-0.2 mL isotonic saline given 24 h apart. One control group of the SCLC-xenografted mice received no treatment (n = 5); the mice in this group were killed after 20 days. The177Lu-DOTA-Tyr3-octreotate-treated SCLC-xenografted mice were divided into five groups that were sacrificed at 1 day (n = 5), 5 days (n = 5), 12 days (n = 4), 20 days (n = 3), and 150 days after treatment or until the tumour had regrown to the original size (n = 4). The GOT1-xenografted mice were divided into two groups untreated (n = 3) and 3 days (n = 3) of treatment. Tumour tissues were dissected, fixed in buffered formalin for 4 to 24 h, and embedded in paraffin. The tumours from each group were cut in serial sections, 4 μm-thick, and subjected to haematoxylin/eosin (H&E) staining, immunohistochemistry, and microscopy to determine cell numbers (tumour and mouse cells) and cell nucleus size.Twenty SCLC-xenografted mice were injected in a lateral tail vein with three fractions of 30 MBqThe dosimetry calculations were based on the pharmacokinetics determined previously in NCI-H69 and GOT1 bearing nude mice ,38. In b177Lu-DOTA-Tyr3-octreotate were stained with the following antibodies: monoclonal mouse anti-human-Ki67 , monoclonal mouse anti-human-Bcl-2 oncoprotein , monoclonal rabbit anti-somatostatin receptor subtype 2 (anti-SSTR2), HPA007264 , monoclonal mouse anti-human wild type-p53 , monoclonal mouse anti-human CD133/1 , monoclonal anti-human thyroid transcription factor (TTF-1) , polyclonal anti-human synaptophysin , and polyclonal anti-human chromogranin-A .Tumour sections from control mice (no treatment) and from mice treated withHybridoma cell line that produced the monoclonal TS1 antibody, which reacted with keratin 8 (K8), was cultured as described previously . The TS1H&E staining was performed on a few selected slides from each group to study the morphology of the cells and determine the presence of necrosis, fibrosis, and apoptosis. Necrosis was defined as an area stained with haematoxylin that originated from proteins released from the cytoplasm of dead cells. Fibrosis was defined as the presence of proliferating fibroblast-like cells, often with an apoptotic appearance. Apoptotic cells were defined as cells with shrunken cytoplasms and nuclear chromatin condensation or intense DNA staining in condensed pyknotic nuclei.The epitope retrieval procedure for commercial antibodies was performed as described by the manufacturer. For TS1, we performed heat-induced epitope retrieval in 10 mM Tris/10 mM EDTA-buffer, pH 9.0. For immunohistochemical staining, we used 5 μg/ml TS1 and the dilution recommended by the manufacturers for the commercial antibodies. A minimum of one section from three to five tumours from each group was analysed immunohistochemically with TS1, anti-Ki67, anti-Bcl-2. Sections were also analysed with each antibody, as follows: anti-somatostatin receptor subtype 2 antibody, one to three sections from each group; anti-human-p53, three sections to confirm negativity; anti-human CD133/1, one to two sections from each group; anti-human TTF-1, three sections to confirm negativity; anti-synaptophysin, five sections; and anti-chromogranin-A, five sections. Appropriate positive controls were used for all antibodies. The immunohistochemistry was performed with the standard method of horseradish peroxidise and diamonobenzidine (DAB) in a Dako autostainer with the EnVision standard reagents. After the immunostaining, the nuclei were counterstained with Mayer's haematoxylin, dehydrated, and mounted.177Lu-DOTA-Tyr3-octreotate in the SCLC xenograft, the ratio of tumour versus mouse cells was calculated and the sizes of cell nuclei were measured in a total of one to two sections from each of two to five tumours in the different treatment groups. A grid with 130 cross-points was placed over an area of 200 × 154 μm in the centre of each section. Tumour cells were defined as cells that stained positively for Ki67 or Bcl-2, cells with abnormal, angular, or polymorphic morphologies, or cells with a hyperchromatic nucleus and reduced cytoplasm. Those located within the cross-points of the grid were counted. Normal mouse cells were defined as cells that did not stain for Ki67 or Bcl-2 and had morphologies of normal mouse fibroblasts, adipose, and lymphoid cells.To evaluate the overall effect ofIn ten tumour cells, we measured the size of the nucleus, or the area with nuclear chromatin, in the cells in the centre of the section. We selected one to three sections from two to four tumours of each group, and systematically measured nuclei within the cross-points of the 200 × 154 μm grid, starting at the top left corner and continuing to the right bottom corner of the grid. The nucleus diameter measurement was performed in two directions and the value was expressed as the mean value and standard deviation. We used a Zeiss AXIO imager A1 microscope equipped with a Zeiss Axiocam. The calculations for the positively and negatively stained cells and for the nucleus size were confirmed with the AXIO Vision programme, release 4.7 (data not shown).t tests and the significance level was set at P < 0.05.The K8 and Ki67 expression levels in treated and untreated NCI-H69 SCLC and GOT 1 midgut tumour xenograft sections were quantified by analysing DAB staining with the ImageJ programme developed by the National Institute of Health . The immWithin each of the seven biopsies, two to nine 320 × 250 μm areas with tumour cells, necrotic tissue, and high TS1 staining were captured at a ×40 magnification and a resolution of 6.24 pixels/μm. These images were analysed with ImageJ plugin ImmunoRatio to calculate the percentage of DAB staining.The injection of three times 30 MBq resulted in different mean absorbed doses to the H69 tumour tissue. For the mice sacrificed 1, 5, 12, 20, or 150 days after last injection the mean absorbed dose was estimated to be 13, 32, 51, 59, and 64 Gy, respectively. The mean absorbed dose to the GOT1 tumours was estimated to be 27 Gy.Histologically, the untreated control, 1-day-treated and regrown tumours showed the classical SCLC cell morphology with abnormal, angular, polymorphic, hyperchromatic nuclei, and scant cytoplasm. Large necrotic regions were present in the untreated, 1- and 5-day-treated tumours. After 12 and 20 days of treatment, the larger necrotic regions were replaced by fibrotic tissue to a large extent. H&E staining showed that the control tumours and regrown tumours were similar, but the regrown tumours had slightly larger areas of fibrosis. Compared to the untreated controls, treated tumours, particularly after 5 days, had higher numbers of small (50 × 50 μm) necrotic regions. Relatively few cells exhibited an apoptotic appearance with a shrunken cytoplasm and intensely stained DNA in condensed pyknotic nuclei. In tumours treated for 5 days, a slightly higher number of apoptotic cells with intense hematoxylin staining were observed between the necrotic areas and viable cells.177Lu-DOTA-Tyr3-octreotate  and 38% after 12 days . After 20 days of treatment, the mean value in the two analysed tumours was 40% , the outer membranes of mitochondria, and in the nuclear envelope. In this study, Bcl-2 expression was located in the tumour cell cytoplasm (ER and/or mitochondria) of untreated tumours. During treatment withm Figure .177Lu-DOTA-Tyr3-octreotate had a dramatic effect on the tumour cells. After only 12 days of treatment, a 50% reduction in tumour cell density was observed in three of four tumours, and the fourth tumour showed only a few viable tumour cells at the edge of the tumour ; in the other three specimens, the cell number had reduced to about 50% of that observed in the untreated control. After 20 days of treatment, tumour cells were eliminated in one of three tumours (n = 3). In the other two tumours, the cell density had reduced to one fourth of that observed in the untreated control. It is well known that irradiation of tumour cells can induce several cell-death mechanisms, including apoptosis, necrosis, autophagy, senescence, and mitotic catastrophe [177Lu-DOTA-Tyr3-octreotate treatment. These nuclei had enlarged to about three times the size of the nuclei in the untreated control. This enlargement was sometimes accompanied by the appearance of multinucleated cells. The formation of multinucleated giant cells was most likely a result of mitotic loss or mitotic catastrophe in the cells.For the SCLC cell line H69 tumours, the three times 30 MBq protocol caused minor effects after 1 day, somewhat more pronounced after 5 days, and extensive after 12 and 20 days. After 12 days, we observed a nearly 100% regression (cell number) in one of four tumours  has 88% identity with the Mus musculus somatostatin receptor 2 (NM 009217.3). However, the mRNA levels of a gene do not always correlate to the levels of protein expression. Thus, PCR-based methods may provide more accurate information on the expression of mouse and human SSTR2 mRNA in this type of xenograft system.It has previously been demonstrated that the expression of SSTR2 mRNA was upregulated as a result ofl effect ,26. The 177Lu-DOTA-Tyr3-octreotate treatment observed after 12 and 20 days, only one of four tumours showed 100% regression after 150 days; the recurrent tumours were histologically similar to untreated tumours. This suggested that, in the majority of tumours, a few cells must have escaped radiation treatment and regained tumour cell properties and viability. Resistance to treatment, relapse, and the presence of metastases has been attributed to the presence of resting cancer stem cells. Other studies have identified cells with the stem cell marker, CD-133, in SCLC tumours [By visual examination, the positivity of TS1, anti-Ki67, and anti-Bcl-2 appeared to increase in the remaining viable cells of the treated tumours Figure , and 4.  tumours . HoweverViable tumour cells could be detected within in the outermost regions of large necrotic areas in untreated Figure , 1- and 177Lu-DOTA-Tyr3-octreotate treatment in both the NCI-H69 SCLC and midgut GOT1 xenografts. The optimal time point to administrate TS1 should be when the level of smaller necrotic regions is high. In vivo diffusion capability of TS1 have been demonstrated earlier [177Lu-DOTA-Tyr3-octreotate treatment as well as the outermost areas of larger necrotic regions can probably be reached by diffusion of the TS1 antibody over short distances in vivo.We detected an increased exposure of the TS1 target, K8 in particular in small necrotic areas in tumours after 5 and 3 days of earlier ,31,50,51High affinity interaction between an antibody and its antigen not only enables a longer antibody retention in tumours it can also reduce the distance an antibody will diffuse within a tumour, therefore comparison of intact and fragments of TS1 should be studied in the future. Recently, monovalent and divalent scFv forms of TS1 were generated that had better diffusion capabilities . It was In vivo, one would not expect TS1 to target intact viable normal cells or tumour cells. However, necrotic regions present in the biopsies were generally small, and close to fast dividing tumour cells. These facts might facilitate contact between TS1 and K8 and increase the distribution of TS1 close to viable tumour cells in vivo. Image analysis of the biopsies demonstrated that five out of seven patient biopsies in the analysed regions expressed K8 to an equal or higher level than that observed in untreated SCLC xenografts. In addition, a pre-radiation treatment with177Lu-DOTA-Tyr3-octreotate would most likely further increase the level of exposed K8 in the tumour.In the human biopsies of primary bronchial small-cell carcinoma, tumour cells showed high positivity for SSTR2 and normal cells tended to be negative. In contrast, these biopsies showed positivity for TS1 in normal lung epithelial cells, tumour cells, and in necrotic regions between fast growing viable tumour cells. 177Lu-DOTA-Tyr3-octreotate, when administered in optimised, fractionated doses in combination with other treatments, could be an alternative treatment also for SCLC.There is an urgent need for new improved treatment modalities for SCLC. The results presented in this study are relevant to the understanding of potential pathways for development within this field. Our results on the tumour xenografts suggested that177Lu-DOTA-Tyr3-octreotate treatment increased the TS1 accumulation in vitro in SCLC and midgut xenografts. This indicated that the combination of177Lu-DOTA-Tyr3-octreotate and radiolabelled TS1 could be valuable to investigated further since it holds promise as a future treatment for SCLC.Our results demonstrated thatER: endoplasmic reticulum; K18: keratin 18; K8: keratin 8; SCLC: small-cell lung carcinoma; SSTR2: somatostatin receptor subtype 2The authors declare that they have no competing interests.AE and PB initiated the idea to use TS1 in the SCLC and GOT1 xenografts. AE performed the immunohistochemistry and the interpretation of the histological results, calculation of mouse and tumour cells and drafted the manuscript. PB and EFA conceived the animal study, and helped to draft the manuscript. TS performed the study with biopsies from primary small-cell lung carcinoma and was involved in the interpretation of the histological result as well as reading and approval of the final manuscript.


Sir,Endometriosis is a common disease characterized by ectopic growth of endometrial tissue that responds to hormonal stimulation. It primarily affects women in the reproductive age group with mixed symptomatology. Cytological features of endometrial cells in peritoneal fluid are not well described, which we wish to highlight.A 30-year-old woman presented with lower abdominal pain and dysmenorrhoea of 1 year duration. On examination, she had mild ascites. Bilateral cysts were seen near the fallopian tube on laparoscopy. Diagnostic considerations included neoplastic process or pelvic endometriosis. A peritoneal wash was performed and 100 mL of haemorrhagic fluid was sent for cytological examination. On microscopy, the fluid showed the presence of endometrial cells. The glandular epithelial cells were arranged in variably sized spheres in which the periphery showed epithelial cells, along the border and as a honeycombed sheet, with a cluster of stromal cells in the centre having hyperchromatic nuclei, scant cytoplasm and indistinct cytoplasmic borders . There wThe utility of peritoneal wash cytology for diagnosis of endometriosis has been reported.2 In most1632


Here we investigate the relationship between changes in glioma metabolism by proton magnetic resonance spectroscopic imaging (1H-MRSI. Needle biopsies were performed under the guidance of neuronavigation prior to craniotomy. Intraoperative magnetic resonance imaging (MRI) was performed to evaluate the accuracy of sampling. Haematoxylin and eosin, and immunohistochemical staining with IDH1, MIB-1, p53, CD34 and glial fibrillary acidic protein (GFAP) antibodies were performed on all samples. Logistic regression analysis was used to determine the relationship between Cho/NAA and MIB-1, p53, CD34, and the degree of tumour infiltration. The clinical threshold ratio distinguishing tumour tissue in high-grade (grades III and IV) glioma (HGG) and low-grade (grade II) glioma (LGG) was calculated.Eighteen patients with different grades of glioma were examined using In HGG, higher Cho/NAA ratios were associated with a greater probability of higher MIB-1 counts, stronger CD34 expression, and tumour infiltration. Ratio threshold values of 0.5, 1.0, 1.5 and 2.0 appeared to predict the specimens containing the tumour with respective probabilities of 0.38, 0.60, 0.79, 0.90 in HGG and 0.16, 0.39, 0.67, 0.87 in LGG.1H-MRSI to guide the extent of resection has the potential to improve the clinical outcome of glioma surgery.HGG and LGG exhibit different spectroscopic patterns. Using  The technique, based on chemical shift imaging (CSI), facilitates characterisation of the tumour and surrounding normal brain tissue by determining the metabolic ratios of choline-containing compounds (Cho), N-acetyl-aspartate (NAA) and creatine (Cr) that are detected in the spectra ) environment. The IDs of the biopsy targets were entered followed successively by the rda and neuronavigation files Fig. . NeuronaBiopsies were conducted within the dedicated intraoperative MRI integrated neurosurgical suite . The neuronavigational MRI data sets with the labelled marks were transferred to the planning workstation of the surgical navigation system  and image fusion was performed, with other MRI sequences that had been obtained beforehand, using StealthMerge software  Fig. .1H-MRSI. Each sampling location was recorded during the course of their extraction by using the screen save feature of the surgical navigation system and was renamed. Intraoperative MR scanning was subsequently performed to confirm the accuracy of the needle biopsy  which was tracked by the navigation system. A burr hole was made by referring to the trajectory made beforehand, and a skull-mounted trajectory guide was used to align and stabilise the biopsy needle before passing it through the brain to obtain samples Fig. . The opepsy Fig. .Each biopsy sample was fixed in 10 % formalin and sent to the Neuropathology Department of Huashan Hospital. The samples were embedded in paraffin and cut into sections for histopathological assessment.Haematoxylin and eosin (H & E)-stained sections of all tumours were reviewed under a light microscope  by two blinded neuropathologists (H.C. and Y.W.) and categorised according to the fourth edition of the World Health Organisation Classification of Tumours of the Central Nervous System (2007).Immunohistochemistry was performed using the EnVision method with diaminobenzidine (DAB) as a chromogen. The primary antibodies were: IDH1 R132H , MIB-1 , p53 , CD34  and GFAP   representing tumour cell proliferation was calculated as the percentage of positively stained nuclei and was classified as: strong (MIB-1 ≥ 10 %), moderate (5 ≤ MIB-1 <10 %), mild (1 ≤ MIB-1 <5 %), or negative (MIB-1 <1 %).Evaluation of the p53, CD34 and GFAP expression was divided into four groups according to the percentage of positively stained tumour cells: strong , moderate , mild , and negative (−).Observation of cell morphology (magnification ×200) was performed at five random fields of concentrated tumour cells. Information was collected on cell density, nuclear abnormalities and mitosis. Tumour infiltration into each biopsy specimen Fig.  was cateAll statistical analyses were undertaken using SAS version 9.2 software. Kruskal-Wallis test was performed to compare the Cho/NAA ratios of biopsy samples from different regions. The relationship between dependent variables  and independent variable (Cho/NAA) was calculated using Logistic Regression (SAS PROC LOGISTIC). Specimens were categorised into tumour or non-tumour tissue to further define HGG and LGG specimens with tumour infiltration. The probability of HGG and LGG specimens containing a tumour cells is calculated using logistic regression functions when Cho/NAA is 0.5, 1.0 1.5, 2.0.P values ≤ 0.05 were considered statistically significant for all tests.A total of 91 biopsy specimen loci were labelled and documented on the 3D neuronavigation MRI data sets Fig. . The hisP < 0.0001, Kruskal-Wallis test).The number and percentage of biopsy samples at different biopsy locations and histological classification of the individual biopsy specimens in HGG and LGG are summarised in Tables P < 0.0001, Kruskal-Wallis test).Thirty–one biopsy samples collected from seven patients with LGG were composed of 20 LGG samples and 11 samples without neoplastic tissue. One biopsy sample that did not contain any tumour cells came from the TC of a grade II glioma. The Cho/NAA ratio from these samples at TC, IPR, DPR were 2.375, 1.13, 0.900 respectively. The difference of Cho/NAA between these locations was also statistically significant or the Cho/Cr ratio [MRS findings have been shown be closely related to histological features of glioma cells and can be used in tumour differentiation, grading, follow-up and radiotherapy planning , 21, 27.Cr ratio .P = 0.001) and stronger CD34 expression (P = 0.0155) in HGG, no correlation was found in LGG. expression.It has been reported that MIB-1 may be the best index for predicting the potential of tumour proliferation, tumour grade and outcome . CD34 ha1H-MRSI was used to avoid the limitations related to single-voxel 1H-MRSI and was able to detect the consecutive metabolism change of gliomas. It has also been proposed that the invasive and aggressive nature of malignant astrocytomas may be related to p53 abnormalities [Other workers have demonstrated a linear correlation between Cho and MIB-1 in gliomas that showed a homogeneous pattern with MRI scanning , 30. Likmalities . However1H-MRSI to identify a CNI that predicted tumour tissue and ruled out normal tissues [Many studies have focused on pursuing cut-off value of different metabolite ratios and have used these ratios in an attempt to contour gliomas. McKnight and collaborators conducted a study with  tissues . They co tissues  found th tissues  failed tIn our study, the metabolite ratio of Cho/NAA was found to predict whether or not a spectroscopic voxel contained tumour cells. We showed that specimens from patients with HGG contained tumour cells with a probability of 0.60 when the Cho/NAA ratio was 1.0 and with a probability of 0.67 in patients with LGG using a Cho/NAA ratio of 1.5. Different threshold values should, therefore, be adopted to delineate tumour margins in HGG and LGG. Our Cho/NAA ratio threshold value for distinguishing tumour from non-tumour in HGG was comparable to that reported by Widhalm et al. , who defBoth glioma grade and the location of glioma need to be considered when determining the glioma margin. More extensive resection may be required when the tumour is located in a non-eloquent region and in these cases the boundaries of the glioma resection should be based on a lower Cho/NAA ratio. Caution should be taken when the tumour is located in an eloquent region. For these cases, the resection margins should be based on a higher Cho/NAA ratio, in accordance with the functional MRI or intraoperative neurophysiological monitoring, to avoid the potential neurological deficits. A balanced approach aimed at increasing the extent of resection and decreasing morbidities, will lead to high quality survival.To date, no unified criteria exist for assessing tumour infiltration. Stadlbauer et al.  defined A feature of our study was that the biopsy targets were consecutive, being located in the TC and tumour border (IPR and/or DPR). The use of consecutive biopsy targets is in accordance with the biological infiltrative behaviour of glioma. We showed that the Cho/NAA ratio was correlated with the location of the biopsy. The Cho/NAA ratios at the TC, IPR, DPR were respectively 4.1, 1.04, 0.73 in HGG and 2.375, 1.13, 0.900 in LGG. The differences in Cho/NAA ratios at different locations in HGG and LGG were statistically significant. The probability of IPR, DPR being infiltrated by glioma was respectively 57 % and 20 % with HGG and 25 % and 20 % with LGG. All of HGGs consisted of at least two different grade compositions. Thirteen biopsies from the TC in HGG patients were classified as grade II glioma, one biopsy from the TC of a LGG patient was classified as gliosis indicating that the glioma was profoundly heterogeneous. These findings were in agreement with McKnight et al.’s study . Cell in1H-MRSI information on navigational images due to the different formats of MRI data sets. However, the NAV_biopsy software developed at our laboratory enabled us to successfully label biopsy targets in the navigation sequence without the need for recording the coordinates of each voxel preoperatively. In other studies [In our study we were able to accurately match histological specimens with voxels. It is usually difficult to label the  studies , 23, bio1H-MRS technique used to provide cellular metabolic information is influenced by the cell growth cycle. Cells in the growth stage can be detected by 1H-MRS but cells in quiescent stage cannot be detected due to their slow metabolism, which means that 1H-MRSI is not sensitive to metabolically silent areas. The voxel volumes in our study were large (7.5 × 7.5 × 15 mm) in comparison to the volumes of the specimen (1.0 × 1.0 × 3.0 mm). Smaller voxels would have allowed the 1H-MRS to reflect the specimen exactly. However, smaller voxels require a long acquisition time which is unbearable for some patients. Using higher field strengths may solve this problem in future studies.Limitations of our study should, however, be noted. The 1H-MRSI can better reflect glioma metabolism and delineate the glioma boundaries. HGG and LGG exhibited different spectroscopic patterns and had different threshold values that can predict the probability of a specimen containing a tumour. Using 1H-MRSI to guide the extent of resection has the potential to significantly improve the rate of gross total resection and subsequently prolong the overall survival time.The preliminary results of our study demonstrated that Cho/NAA is closely related to MIB-1, CD34 and tumour infiltration in HGG. Compared with conventional MRI,


PG545 is a heparan sulfate (HS) mimetic that inhibits tumour angiogenesis by sequestering angiogenic growth factors in the extracellular matrix (ECM), thus limiting subsequent binding to receptors. Importantly, PG545 also inhibits heparanase, the only endoglycosidase which cleaves HS chains in the ECM. The aim of the study was to assess PG545 in various solid tumour and metastasis models.in vivo angiogenesis, solid tumour and metastasis models. Pharmacokinetic (PK) data were also generated in tumour-bearing mice to gain an understanding of optimal dosing schedules and regimens.The anti-angiogenic, anti-tumour and anti-metastatic properties of PG545 were assessed using in vivo and induce anti-tumour or anti-metastatic effects in murine models of breast, prostate, liver, lung, colon, head and neck cancers and melanoma. Enhanced anti-tumour activity was also noted when used in combination with sorafenib in a liver cancer model. PK data revealed that the half-life of PG545 was relatively long, with pharmacologically relevant concentrations of radiolabeled PG545 observed in liver tumours.PG545 was shown to inhibit angiogenesis PG545 is a new anti-angiogenic clinical candidate for cancer therapy. The anti-metastatic property of PG545, likely due to the inhibition of heparanase, may prove to be a critical attribute as the compound enters phase I clinical trials. Tumour angiogenesis has become a prominent drug target for cancer therapy  and agenβ-glucuronidase heparanase (HPSE) is strongly implicated in cell dissemination associated with tumour metastasis  from the extracellular matrix (ECM) (Cleavage of HS by the endo-ix (ECM) . The cliKi=6 nM) and a potent angiogenesis inhibitor (⩽1 μM) using endothelial cell assays in vitro and the ex vivo the rat aortic ring assay. Moreover, PG545 was found to have mild anti-coagulant properties using the activated partial thromboplastin time test and the Heptest compared with PI-88 or earlier PG500 series compounds  profile to support less frequent dosing compared with other HS mimetics. An open-label, single centre phase I study of the safety and tolerability of PG545 in patients with advanced solid tumours commenced in late 2010 .We report here the pharmacological profile of PG545, a synthetic, fully sulfated HS mimetic. We have previously demonstrated that PG545 is a potent heparanase inhibitor -3H]-cholesterol , were manufactured at Progen Pharmaceuticals Ltd and PharmaSynth . The tyrosine kinase inhibitor (TKI) sorafenib is an inhibitor of Raf-1 and other kinases involved in neovascularisation and tumour progression , pH 7.2 for in vivo studies.PG545 is a fully sulfated tetrasaccharide functionalised with a cholestanyl aglycon  designed−1 penicillin (Invitrogen), 100 μg ml−1 streptomycin , 1 mM sodium pyruvate (for LL/2 and HepG2 only: Invitrogen), 25 mM HEPES (HepG2: Invitrogen) and 0.4 mg ml−1 hydrocortisone .LL/2, Hep3b2.1-7 and HT-29 tumour cell lines were obtained from the American Type Culture Collection and working stocks did not exceed four passages. MDA-MB-231, PC3, Cal27 and HepG2 were from laboratory stocks at the Diamantina Institute, Brisbane, Australia. Identity was verified by STR analysis conducted by Cell Bank Australia  in February 2010. B16F0 was a gift from Dr Glen Boyle . Hep3b2.1-7 and PC3 were cultured in RPMI-1640  while MDA-MB-231, B16F0, HT-29, HepG2, Cal27 and LL/2 were cultured using DMEM (Invitrogen) and supplemented with 10% fetal bovine serum , 100 IU mlAthymic (nude), SCID, FvB, BALB/c and C57Bl6 mice were obtained from the Animal Resources Centre . All animal studies were approved by the University of Queensland or the University of Adelaide Animal Ethics Committees. During the studies, the care and use of animals was conducted in accordance with the principles outlined in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 7th Edition, 2004 . For the HT-29 study, the protocol was approved by the Institutional Animal Care and Use Committee and the study was performed at the AAALAC certified animal facility in Pharma Legacy .in vivo was evaluated using the Angiosponge model in which subcutaneously implanted, agarose-captured FGF2 gelfoam sterile sponges  induces vascularisation over a 10-day period responsive to angiogenesis inhibition as determined by the number of vessels with identifiable lumen that stained positive for CD31/PECAM-1, an antigen present specifically on endothelial cells (Angiogenesis n=10) began 14 days after inoculation of 2.5 × 106 cells. Mice received PG545 (or [3H]-PG545 for the efficacy/PK study) using either a once- or twice-weekly dosing schedule. To initiate tumour xenografts, MDA-MB-231, PC3, Cal27 and HepG2 cells were injected s.c. into nude mice (n=8) at 4, 5, 2 and 2 × 107 cells ml−1, respectively. PG545 was injected s.c. in a volume of 100–200 μl PBS once the average tumour volume was ∼100–200 mm3. In the B16F0 lung metastasis model, 2 × 105 cells were injected via the tail vein of C57Bl6 mice (n=8). In the syngeneic LL/2 model, single dose or weekly treatments with PG545 at 20 or 40 mg kg−1 and sorafenib daily at 60 mg kg−1 commenced on the day of inoculation with 2 × 106 cells. Surface lung macrometastases were counted for each group (n=9) upon study termination. In the intrasplenic HT-29 xenograft model, 1 × 106 cells in 100 μl were injected into the spleen. The next day, PG545 (20 mg kg−1) was administered to mice (n=12) each week (qw) over 8 weeks and surface liver and colon metastatic nodules were counted for each group (n=12) upon study termination. Solid tumours were measured in two dimensions (length and width) and the tumour volume (cm3) was calculated using the equation: V=length × width2 × π/6. The anti-tumour activity was expressed as percent tumour growth inhibition (%TGI). Further details are available in In the orthotopic Hep3b2.1-7 human liver cancer model, treatment of mice  collected at 2, 4, 8, 24, 48, 72 and 96 h post treatment were determined as outlined in Cmax (maximum plasma concentration), Tmax (time of maximum plasma concentration), kelim , t½  and AUC (area under the concentration vs time curve) were derived from the blood [3H]-PG545 or plasma PG545 concentration vs time data using model independent methods. Analysis of the tumour [3H]-PG545 concentration vs time data required use of a more physiologically based PK model such that tumour concentrations were estimated from the tumour mass being perfused with blood containing [3H]-PG545 and where the blood [3H]-PG545 concentration changed over time.Levels of -PG545 administered s.c. every 96 h for a total of three doses. The concentration of PG545 was assessed in both blood and tumour samples. The PK profile in blood indicated that the mean elimination half-life (t½) of [3H]-PG545 was >50 h for mice administered up to three s.c. bolus doses at 96 h intervals , 48 h (two doses) and ⩾96 h (three doses) (3H]-PG545 from the bloodstream into the tumour (t½b), derived from the mean blood and tumour concentration vs time data, was in the range 12.8–16.5 h. The corresponding estimated mean t½ for return of [3H]-PG545 from the tumour into the bloodstream (t½t), derived from the mean blood and tumour concentration vs time data, was in the range 15.7–16.3 h . This equates to a tumour:liver ratio of 0.4 and a tumour:kidney ratio of 1.3, 12 h after a single dose, suggesting good distribution into tumour tissue compared with other organs. As a comparison, gefitinib  has a tumour:liver ratio of 0.15 or 0.18 and a tumour:kidney ratio of 0.35 or 0.56, 2 h or 8 h, respectively, after a single dose in LoVo tumour xenografts  . Using a7–16.3 h . The Cma−1 once-weekly (77%) or 20 mg kg−1 twice-weekly (71%) with 30 mg kg−1 groups inhibiting tumour growth by 69%  or 59% . Body weight loss at the end of the study was 4% for 20 mg kg−1 once-weekly, 6% for 20 mg kg−1 twice-weekly, 11% for 30 mg kg−1 once-weekly and 10% for 30 mg kg−1 twice-weekly (data not shown). Employing the 20 mg kg−1 dose only in subsequent models, the differences noted between once- and twice-weekly schedules were not significant in the PC3 or HepG2 models. For example, in the PC3 model, once-weekly dosing led to a TGI of 74% while twice-weekly dosing resulted in a TGI of 83% . However, in the HepG2 model  led to body weight loss between 2% and 4% (data not shown). PG545 also significantly reduced primary tumour growth in the HT-29 model (50) using HUVEC (78 μM), B16 (>200 μM) or HepG2 cells (>100 μM), indicated that PG545 is not cytotoxic in vitro (data not shown).Further investigation of once- or twice-weekly s.c. dosing schedules was performed in a variety of xenograft or syngeneic cancer models . Tumour I of 83% . AlthougG2 model , the oncI of 57% . In both29 model . The conTwice-weekly injections of PG545 significantly inhibited the formation of metastases in the B16F0 experimental lung metastasis model, when administered either before or on the day of tumour cell inoculation . When ad−1 group and 96% inhibition in the 20 mg kg−1 once-weekly group  provided another model to investigate the effect of PG545 on spontaneous metastasis. PG545 was administered at weekly intervals or in some cases, as a single injection from the day of tumour inoculation. The body weight nadir during the study constituted a loss of 5% (data not shown). In addition to significant inhibition of primary tumour growth , PG545 sly group . However μg ml−1  and PK acompound . The difex vivo angiogenesis  and did not lead to significant body weight loss in other species during toxicity studies (data not shown). The absence of bruising in PG545-treated nude mice illustrates the mild anti-coagulant properties of PG545 (The AngioSponge model highlighted that daily and twice-weekly dosing with PG545 induced a potent anti-angiogenic effect mimetics . The bodt½) in blood (>50 h) was observed. The concentrations of [3H]-PG545 measured within the liver tumours of mice confirmed entry into tumour tissue. A one-compartment PK model found each of the estimated parameter values was internally consistent following administration of one, two or three doses . A relatively long half-life . In the spontaneously metastasising Lewis Lung Carcinoma model, PG545 significantly reduced the number of metastases and PK data estimated the efficacious plasma concentration range starts at ∼20 μg ml−1. This potent anti-metastatic effect of PG545 may differentiate it from other angiogenesis inhibitors, which have been associated with an acceleration of metastasis under certain circumstances (We next tested the anti-metastatic activity of PG545 in three murine models of metastasis. In the B16 melanoma model, short-term therapy with PG545 was initiated either before or immediately after i.v. tumour inoculation of B16 cells and confirmed that both schedules significantly inhibited the development of lung metastases. In the spontaneously metastasising HT-29 colon model, complete abrogation of liver metastases was achieved using a weekly regimen of 20 mg kgIn conclusion, we have clearly demonstrated the anti-tumour and anti-metastatic properties of PG545, which to our knowledge is the first HS mimetic that is a single molecular entity to emerge as a clinical candidate. In addition to its anti-angiogenic properties, it is the anti-heparanase/anti-metastatic activity that may differentiate PG545 from other anti-angiogenic agents. Particularly in light of recent preclinical findings relating primarily to anti-VEGF therapies, this property may become a critical attribute as PG545 progresses towards the clinic.


Tumour vasculature acts as an essential lifeline for tumour progression and facilitates metastatic spread. Novel vascular targeting strategies aiming to sustain vascular shutdown could potentially induce substantial damage, resulting in a significant tumour growth delay. We investigated the combination of two novel complementary vascular targeting agents with radiation therapy in a strategy aiming to sustain vascular disruption. Experiments were carried out with delta-like ligand 4 (Dll4) blockade (angiogenesis deregulator) treatment administered in combination with a radiation-based vascular destruction treatment in a highly aggressive well-perfused colon cancer tumour line implanted in female athymic nude mice. Tumours were treated with permutations of radiation, ultrasound-stimulated microbubbles (USMB) and Dll4 monoclonal antibody (mAb). Tumour vascular response was assessed with three-dimensional power Doppler ultrasound to measure active flow and immunohistochemistry. Tumour response was assessed with histochemical assays and longitudinal measurements of tumour volume. Our results suggest a significant tumour response in animals treated with USMB combined with radiation, and Dll4 mAb, leading to a synergistic tumour growth delay of up to 24 days. This is likely linked to rapid cell death within the tumour and a sustained tumour vascular shutdown. We conclude that the triple combination treatments cause a vascular shutdown followed by a sustained inhibition of angiogenesis and tumour cell death, leading to a rapid tumour vascular-based ‘collapse’ and a significant tumour growth delay. Vascular development in tumours occurs via the release of various endothelial growth factors and angiogenesis regulators , platelet derived growth factor (PDGF), delta-like ligand 4 (Dll4)), leading to neo-vascularization predominantly via a sprouting process reminiscent of embryonic development Tumours require activation of an angiogenic ‘switch’ to grow beyond 1–2 mmA variety of physiological and biological factors stimulate tumour vascular development (i.e. hypoxia). These stimuli act as external queues for angiogenic  molecular signalling. The Notch pathway has been demonstrated to regulate the sprouting of new vessels in tumours et al.asmase-dependent, acute endothelial apoptosis following single large doses of radiation Alongside the numerous pharmacological agents that have been developed to target angiogenesis, emerging biophysical vascular targeting agents, including ultrasound-stimulated microbubbles and gold nanoparticles, have also been demonstrated to increase conventional cancer treatment efficacy In this work, we have investigated Dll4-blockade following combined ultrasound-microbubble and radiation-based vascular destruction in a highly aggressive and well-perfused colon cancer line. Tumours were grown in the hind leg of mice and treated with permutations of radiation, USMB and Dll4 monoclonal antibody (mAb). Animals were imaged using three-dimensional power Doppler ultrasound to investigate changes in tumour perfusion (blood flow) with treatment.Three-dimensional high-frequency power Doppler ultrasound is an ideal imaging modality for the pre-clinical assessment of tumour vasculature in animal models The results indicate that Dll4 blockade followed by USMB and radiation produces a synergistic tumour growth delay greater than when radiation is combined with Dll4 mAb alone. These findings also suggest that USMB and radiation act to acutely shut down tumour vasculature, as in other types of tumours, but that due to the aggressive nature of the colon cancer cell line, a rapid tumour rebound follows. Treatment with Dll4 mAb then deregulates neo-vascularization, leading to minimally functional tumour vasculature and a sustained tumour vascular ‘collapse’. Thus, the combination of DLL4 blockade with USMB and radiation is a novel, highly effective approach to delay tumour growth through biological and biophysical targeting of the tumour vasculature.6) colon cancer cells  were injected subcutaneously into the right hind leg of 6 to 7-week-old female athymic nude mice . All tumours grew for 9–14 days to reach a mean volume of 200 mm3. An average of 5 animals were used per treatment condition for each of the treatment assessment methods .All animal experiments presented in this work were conducted in compliance with internationally recognized guidelines specified in protocols approved by the Sunnybrook Research Institute institutional animal care and use committee. All animal procedures were performed under anaesthesia, as described below, and all efforts were made to minimize suffering. For this study, up to 90 animals were randomly divided into 3 cohorts. Animals in cohort 1 were sacrificed at 24 hours, animals in cohort 2 were sacrificed at 7 days and animals in cohort 3 were kept for tumour growth assessment for up to 30 days. In order to initiate tumour growth, LS174T  no treatment (CTRL), (2) radiation (XRT), (3) Dll4 mAb, (4) XRT and Dll4 mAb, (5) ultrasound-stimulated microbubbles (USMB) combined with XRT, or (6) a triple combination of USMB, XRT and Dll4 mAb. Animals were anesthetised with isofluorane before imaging or for catheter insertion (when receiving USMB treatment). All animals receiving radiation therapy were treated with a single dose of 5 Gy ionizing radiation administered using a Faxitron cabinet irradiator , using X-rays at an energy of 160 kVp, an SSD of 35 cm, and a dose rate of 200 cGy/min. For radiation treatment, the body of each animal was covered with 3 mm thick lead shielding, exposing only the tumour. Animals receiving the Dll4 monoclonal antibody  were administered the mAb twice weekly, starting on the treatment day following imaging, at a dose of 5 mg/kg of body weight. Animals receiving radiation in conjunction with Dll4 mAb, received the initial anti-Dll4 mAb treatment 1 hour after irradiation. Dll4 mAb treatment was continued in animals until sacrifice (at 24 hours or 7 days), or until a tumour volume reached at least double the original volume. A schematic of the treatment delivery and imaging schedule is presented in 9 microbubbles in 90 µL), which is approximately 300-fold the recommended diagnostic imaging dose of 10 ul/kg For animals treated with USMB, Definity microbubbles  were prepared using a Vialmix device  and administered through a tail vein catheter at a volume concentration of 3% v/v (1.08×1024 Hours/VI0 Hours)×100) –100) or ((VI7 Days/VI0 Hours) ×100) –100).All animals were imaged with three-dimensional high-frequency ultrasound immediately before treatment and 24 hours after treatment. Data was acquired using a Vevo 770 high-frequency ultrasound-imaging device  with a 30 MHz center frequency transducer . A motorized scan stage  was used to acquire 3D B-mode images and power Doppler flow data, at a step size of 0.2 mm. Power Doppler data was collected with the following settings: a clutter-filter cut-off of 1–2 mm/s, a scan speed of 1 mm/s, a pulse repetition frequency of 5 KHz, a power Doppler gain of 20 dB and a frame rate lesser than 10 fps. Power Doppler image analysis was conducted using in-house developed software in MATLAB . The vascularity index (VI) was computed from power Doppler images by obtaining the volume of all coloured objects (coloured pixels) over the volume of the selected ROIs. A relative vascularity index was used to assess overall vascular response to therapy, computed as follows: ((VIin situ end labelling (ISEL) stain as a cell death marker, and cluster of differentiation (CD31)  to evaluate vascular density. Cell death was quantitatively evaluated for each of the tumour ISEL-stained cross-sections. This was carried-out using custom MATLAB  routine, which allows the user to compute the number of brown pixels (ISEL-stained) over the total number of pixels per whole tumour cross-section. The vascular density was also computed from images of CD31 stained tumour cross sections. For each cross section, 4 fields of view (FOV) were analyzed at an objective lens magnification of 10X to compute the density of vasculature. This was carried out using MATLAB  to distinguish the brown stained regions  in the tumour from the background stain (other cells).Mice were sacrificed 24 hours, and in another cohort, at 7 days after treatment for histology and immunohistochemistry. Cross sections of tumour xenografts were stained with hematoxylin & eosin (H&E) to evaluate necrosis following therapy. Tumour xenograft cross-sections were also stained for DNA breaks using 2/2). Tumour volumes were normalized to their starting volume and tumour growth delay was calculated by subtracting the average number of days for treatment groups to reach two times their starting volume from that of the control group.Tumour growth was assessed in an average of 5 animals per treatment condition. Tumour volume was determined by calliper measurements performed every 2–4 days and calculated by using the modified ellipsoidal formula . Each treatment condition was compared directly to the 0 Gy control condition. In addition, p values from statistical tests of different permutations have been summarized in We conducted experiments to assess the efficacy of vascular targeting strategies where USMB, radiation and anti-Dll4 treatments were combined to disrupt tumour vasculature and sustain vascular effects in order to maximize overall tumour response . Tumour Power Doppler and cell death results were complemented with results from CD31 staining. Representative CD31 immunohistochemistry is shown in In this study, we set out to investigate the efficacy of strategically combining a biophysical, radiation-based, vascular disrupting treatment followed by an angiogenesis-deregulating pharmacological agent. It was posited that this would optimize treatment response. In order to maximize vascular destruction, ultrasound-stimulated microbubble therapy was used to first radiosensitize tumour endothelial cells, followed by a single dose of ionizing radiation (XRT) to induce rapid vascular effects. Dll4 mAb was subsequently administered as a ‘maintenance’ therapy. An illustration of posited combined USMB, XRT and Dll4 mAb effects on tumour endothelial cells are presented in A decrease in tumour size was observed within 5 days following the triple combination treatment (6), demonstrating an acute response and suggesting a rapid tumour vascular-based ‘collapse’. We observed a power Doppler VI decrease of more than 50% at 24 hours in both XRT+Dll4 mAb (4) and triple combination treatments (6), in comparison to radiation alone (2), where no decrease in the VI was observed . The VI For animals treated with XRT+Dll4 mAb (4) or USMB+XRT+Dll4 mAb (6), a significant and rapid decrease in the vascular index was observed by 24 hours following therapy . HoweverTreatment with Dll4 mAb alone (3) induced a similar response to those previously reported Recent investigations have demonstrated that pre-treating tumours with USMB can radiosensitize endothelial cells to radiation, subsequently increasing the overall response of tumours Following XRT, we noted an increase in the power Doppler vascularity index at 7 days . A similIn summary, the results demonstrated an enhanced tumour response when ultrasound-stimulated microbubble therapy and XRT were combined with Dll4 mAb (6) as a maintenance therapy. Our results clearly demonstrate that a synergistic effect enhances treatment response when USMB, radiation and Dll4 mAb are strategically combined. In the future, we anticipate that a yet greater treatment response can be achieved through the administration of multiple fractionated treatments. Our results are encouraging given the increasing use of single fraction radiation therapy. Such treatments are now commonly used for palliation with the aim of eliciting rapid anti-vascular effects, in turn minimizing bleeding in patients, arresting tumour growth and alleviating pain. The use of USMB and Dll4 mAb in combination with a single radiation fraction may enhance such effects. Results presented in this study also confirm the importance of Dll4 in regulating ongoing tumour angiogenesis and its potential use in combination with other novel cancer therapies. Finally, our results add to the paradigm that tumour blood vessels play an important role in tumour response to therapy and demonstrate a maximal tumour response when tumour vasculature is biophysically disrupted, followed by an angiogenesis deregulating ‘maintenance’ treatment.Figure S1A) Schematic overview of experiments. Tumours were left to grow for 9–14 days. Animals were then imaged and treated accordingly. Follow up imaging took place at 24 hours and 7 days. Animals treated with Dll4 mAb continued receiving the agent until sacrifice. B) Potential model for vascular strategy. While USMB+XRT is likely causing a rapid vascular shutdown, the aggressive nature of the cell line is inducing a rapid vascular rebound. In contrast, Dll4 mAb following USMB and XRT blocks functional vascular rebounds for a longer period of time.(TIFF)Click here for additional data file.Figure S2Representative A) H&E, B) ISEL, and C) CD31 stained tumour cross-section images for each of the treatment conditions at low and high magnification. The scale bar represents 2 mm for ISEL and H&E and 100 µm for CD31.(TIFF)Click here for additional data file.Figure S3Representative H&E images of control, Dll4 mAb, XRT+Dll4 mAb, USMB+XRT and USMB+XRT+Dll4 mAb at tumour growth assay end point . Persisting tumour heterogeneity is observed in the triple combination H&E stained tumour cross-section. The scale bar represents 2 mm.(TIFF)Click here for additional data file.Table S1P-value summary for all quantified 24 hours ISEL staining from all treatment conditions.(DOCX)Click here for additional data file.Table S2P-value summary for all quantified 7 days ISEL staining from all treatment conditions.(DOCX)Click here for additional data file.Table S3P-value summary for all quantified growth delay from all treatment conditions.(DOCX)Click here for additional data file.Table S4P-value summary for all quantified 24 hours VI values from all treatment conditions.(DOCX)Click here for additional data file.Table S5P-value summary for all quantified 7 days VI value from all treatment conditions.(DOCX)Click here for additional data file.Table S6P-value summary for all quantified 24 hours CD31 staining from all treatment conditions.(DOCX)Click here for additional data file.Table S7P-value summary for all quantified 7 days CD31 staining from all treatment conditions.(DOCX)Click here for additional data file.


Spatial relationships between key features  are clearly visualized in volume renderings. Invasive tumour burden can be underestimated using conventional pathology, compared to the volumetric technique . Tumour volume approximated from 2D measurements , assuming elliptical geometry, was seen to overestimate volume compared to the 3D volumetric calculation .Tumour size, most commonly measured by maximum linear extent, remains a strong predictor of survival in breast cancer. Tumour volume, proportional to the number of tumour cells, may be a more accurate surrogate for size. We describe a novel “3D pathology volumetric technique” for lumpectomies and compare it with 2D measurements. Volume renderings and total tumour volume are computed from digitized whole-mount serial sections using custom software tools. Results are presented for two lumpectomy specimens selected for tumour features which may challenge accurate measurement of tumour burden with conventional, sampling-based pathology: (1) an infiltrative pattern admixed with normal breast elements; (2) a localized invasive mass separated from the  Tumour size is a commonly used predictor of survival in breast cancer and correlates strongly with lymph node involvement –5. Tumou in situ and invasive disease.Here we describe a novel methodology for measuring tumour volume in lumpectomies, referred to as the 3D pathology volumetric technique. For proof of concept, we demonstrate the technique using two lumpectomy specimens, comparing volumes as measured from serial whole-mount sections versus simulated conventional, sampling-based pathology. Serial, whole-mount sections are produced utilizing “3D pathology” techniques and then digitized . ConformTumour volume may provide a more accurate representation of size, because tumour is a 3D entity. Volume can vary significantly among tumours which have the same maximum linear extent. A simple ellipsoid model has been used to approximate volume using maximum linear extent and has been shown to provide a more accurate assessment of the volume of breast tumours compared to modelling the tumour as a sphere; in a retrospective study of 165 tumours measuring 2.5 cm or less, the largest diameters in anterior-posterior (AP), medial-lateral (ML), and superior-inferior (SI) dimensions were distinct in 96.4% of the cases . BiologiTo date there are no clear data supporting the superiority of tumour volume over maximum linear dimension as a predictor of outcome in breast cancer, although current pathology guidelines are beginning to incorporate 3D parameters  –12. SomeOne of the impediments to establishing the prognostic value of tumour volume in breast cancer staging stems from inconsistencies in measurement technique, especially for more complex tumour patterns . Diffusely infiltrating tumours exhibit a morphology in which the cancer cells are interspersed with normal epithelial and stromal elements making precise measurement of the volume of tissue occupied by tumour cells difficult. Multifocality occurs in about 30% of breast cancers  and is ain situ carcinoma where the foci are separated by intervening normal breast tissue) and may permit more accurate assessment of tumour burden where conventional sampling is difficult . Studies using whole-mount or large-section pathology techniques confirm accepted prevalence rates for multifocality  and confirm that multifocality is an independent prognostic factor for survival at 10 years [Whole-mount sections have been proposed as a “gold standard” for evaluating multifocality . One case (Case A) is an invasive tumour that infiltrates diffusely, mostly without a desmoplastic reaction or destroying intervening benign breast elements, such that a typical tumour section shows invasive carcinoma admixed with benign breast elements. The other case (Case B) is a localized invasive tumour with foci of in situ carcinoma away from the invasive tumour and separated from it by intervening benign tissue. Case B is, therefore, representative of the challenge inherent in measuring tumour volume for multifocal disease, using conventional techniques. Two lumpectomy cases were selected retrospectively from the whole-mount tumour bank at the Biomarker Imaging Research Laboratory at Sunnybrook Health Sciences Centre. The specimens were initially obtained from the Department of Pathology at Sunnybrook with the approval of the institutional Research Ethics Board, excluding lumpectomies that would be submitted μm thick tissue section was obtained from the top of each block using a sliding microtome . The sections were stained with hematoxylin and eosin (H&E) using manual techniques. Both specimens were prepared and processed using techniques collectively referred to as “3D pathology” , 28. Eacμm as previously determined to optimize tumour detectability and computational feasibility [in situ tumour)  using a pixel spacing of 2 sibility . The sizsibility ; develop tumour) . Using tin situ and invasive disease separately. For each whole-mount image, three binary image arrays were generated to represent the following three features as defined by the digital contouring: in situ disease, invasive disease, and normal tissue, with pixels enclosed in a contour set to intensity values R, G, B = 1, and 0 otherwise. The three binary arrays were stacked and then extruded in the medial-lateral dimensions, by layering duplicate copies of the arrays at every 2 μm, corresponding to the lateral resolution, to fill the 4 mm gap between whole-mount sections. In this way, isotropic 4 mm voxels were preserved in the volume rendering. Tumour volumes were calculated by Riemann summation of all the positive pixels (RGB intensity = 1) in the volume rendering, for in situ and invasive disease separately. For the set of images simulating conventional pathology technique, volume was calculated similarly by multiplying the sampled tumour area by the thickness of 4 mm. For volume calculations and visualization, the  .xml files were imported into MATLAB (MATLAB7.11.0.584 (R2010b); Math Works Inc.; Natick, MA, USA), to generate a volume rendering for each case, and to calculate tumour volumes, for ellipsoid = 1/6π ML·AP·SI. The maximum linear extent in ML, AP, and SI dimensions was measured from the whole-mount sections.Finally, tumour volume was estimated from 2D measurements of maximum linear extent in the ML, AP, and SI dimensions using the following ellipsoidal approximation: VolumeFor Case A, the set of serial whole-mount sections which contain tumour is shown in Tumour volume, calculated from the full renderings as well as from the locations corresponding to conventional sampling, are compared in The 3D pathology volumetric technique facilitates visualization of spatial relationships within lumpectomies and may provide a more accurate surrogate for tumour burden. It is seen from Case A that when the tumour infiltrates without eliciting a desmoplastic reaction and without destroying the normal benign breast elements, the conventional 2D measurements using maximum extent in the three dimensions  may overestimate tumour burden. If the tumour is approximated by an ellipsoid, then using these three measurements the tumour volume is overestimated by a factor of approximately 7 . Howeverin situ component that is present away from the tumour can be readily appreciated. Similarly, the locations of close or involved margins can be easily identified. Taking the close or involved margins seen in the serial whole-mount section images  . SimilarThe 3D pathology volumetric technique is well suited to validation of noninvasive imaging modalities  which are used to estimate 3D tumour descriptors including volume and surface area. Surface area might also serve as a useful reporter for tumour burden in conjunction with volume. Our methodology lends itself to exploration of other quantitative measures including surface area as a simple extension of the volumetric principle. Using the techniques we describe here, we can obtain precise, volumetric measurements of challenging features  and in future studies the prognostic significance of these presentations can be explored using the volumetric technique. Studies on the prognostic significance of tumour volume with relation to biology are scarce. It has been suggested that tumour volume is inversely related with microvessel density, and that metastasis may, therefore, be an early event . The 3D in situ carcinoma away from the tumour). We have shown that in such cases tumour burden can be significantly underestimated using conventional methods. Conversely, tumour volume can be markedly overestimated, especially for more complex tumour distribution , when simple approximations such as an ellipsoid model are used instead of the volumetric approach. Additional studies to evaluate the predictive value of tumour volume on patient outcome are underway.Using the 3D pathology volumetric technique, tumours are visualized in 3D and spatial relationships between key features  are readily appreciated. The technique also enables calculation of tumour volume, which may be a more accurate representation of tumour burden or size compared to 2D linear measurements. This technique also reduces the errors associated with undersampling in conventional histopathology, which can result in underestimation of tumour burden. The volumetric technique may be particularly useful in cases where accurate representation using conventional tissue samples may be difficult (e.g., infiltration without desmoplastic reaction and tumour admixed with normal epithelium, or localized invasive mass with multifocal


AbstractNidularia balachowskii Bodenheimer, based on the adult female lectotype and paralectotype (here designated), and new material collected from Israel are presented. A key for the identification of first-instar nymphs of Nidularia spp. is offered. Molecular identification of Nidularia balachowskii, using nucleotide sequences from the D2–D3 region of the 28S ribosomal gene, and the mitochondrial Cytochrome Oxidase I (COI) gene, is presented. Morphological and molecular analyses confirm that Nidularia balachowskii is closely related to other species within the Kermesidae. In Israel, this species develops only on Quercus ithaburensis and is univoltine. This is the first detailed report of Nidulariabalachowskii from Israel.Descriptions and illustrations of the adult female and first-instar nymph of the kermesid  Nidularia Targioni-Tozzetti, 1868 is one of ten genera of scale insects within the Kermesidae (Hemiptera: Coccoidea). Three species of Nidularia: Nidularia balachowskii Bodenheimer, 1941, Nidularia japonica Kuwana, 1918 and Nidularia pulvinata , PageBreakhave been recorded so far only from the Palaearctic region  a group of multilocular disc-pores near the base of each antenna; (vi) an anal ring with or without cells and (vii) anal ring with or without setae and absence of: (viii) a spiracular band of quinquelocular disc-pores, (ix) stigmatic spines and (x) ventral microducts.The main synapomorphic characters of kermesid adult females have been summarized by several authors including: Nidularia share morphological and biological characteristics with other species of the Kermesidae. Like Kermes spp., Nidularia spp. are monophagous and develop on oak trees. They are known as ‘gall-like insects’ due to the size and body shape of the convex and sclerotized post-reproductive adult female  as well as on fresh material collected from Israel and Turkey. In addition, the first-instar nymph is described and illustrated for the first time. An identification key for the first-instar nymphs of Nidularia is provided. We also compare some characters of the adult female of Nidularia balachowskii with those of several other species of Kermesidae: Nidularia pulvinata, Kermes roboris (Fourcroy) (type species of Kermes) and Kermes quercus (Linnaeus). In addition, sequences of 28S and COI genes from Nidularia balachowskii and five or six other kermesids species plus some scale insect representatives of other Coccoidea families were compared in order to elucidate the family placement of N. balachowskii. Information on habitat and life history of Nidularia balachowskii in Israel is also provided.Bodenheimer’s original description of t female . His desNidularia balachowskii is based on type material , plus specimens from Israel collected by Bodenheimer and fresh material collected in Israel by us. Populations of Nidularia balachowskii from Quercus ithaburensis trees were studied and specimens were collected between 2010 and 2012 from the following nature reserves in northern Israel: Yehudiya Nature Reserve, Golan Heights ; Horshat Tal Nature Reserve, Upper Galilee ; Alonei Abba Nature Reserve, Lower Galilee . Trees at each reserve were surveyed at least once a month and 150–200 branches (20–25 cm in length) were removed at each visit. The branches were taken back to the laboratory in large plastic bags and examined individually under a stereomicroscope for scale insects. Relevant specimens were slide-mounted for microscope examination using the protocol in Ben-Dov, Nidularia pulvinata  became available from MNHN. Material of Nidularia japonica was not available and comparisons with this species were based on the original description by Nidularia balachowskii from Israel, are deposited in the ICVI, BMNH and MNHN.This redescription of Nidularia balachowskii are generalizations of several specimens, showing the dorsum on the left and the venter on the right, with enlargements of important structures arranged around the main drawing. The enlarged structures are not drawn to the same scale. Terms for morphological features follow chiefly those of Illustrations of the adult female and the first-instar nymph of BMNH -The Natural History Museum, London, U.K.; ICVI - Coccoidea Collection, Department of Entomology, Agricultural Research Organization, Bet Dagan, Israel; MNHN - Museum National d’ Histoire Naturelle, Paris, France; and TAU - Tel Aviv University Insect Collection, Israel.PageBreakAbbreviations of specimen depositories are as follows: Turkey: Lectotype female (ICVI), here designated, and paralectotype female (MNHN), 21 km at road from Mardin to Diyarbakir, on branches and twigs of Quercus sp. (Fagaceae), 13.ii.1939, F.S. Bodenheimer. Additional non-type material from Turkey as follows: Van-Koçet Road  on Quercus sp., 19.vii.2005, B. Kaydan ; Hakkari -Üzümcü Road  on Quercus sp., 15.ix.2005, B. Kaydan ; Van-Hakkari Road  on Quercus sp., 16.ix.2005, B. Kaydan ; Hakkari-Doğan  on Quercus sp., 22.v.2005, B. Kaydan ; Bitlis River  on Quercus sp., 23.vi.2006, B. Kaydan ; Bitlis-Kavakbaşı  on Quercus sp. 30.v.2007, B. Kaydan .Israel: adult female, Daphne Oaks , on Quercus sp., 1.v.1939, F.S. Bodenheimer, (ICVI C:4805). This was the first record of this species from Israel. Additional females and first-instar nymphs, all collected off Quercus ithaburensis by M. Spodek: Yehudiya Nature Reserve, 10.x.2010, 7.xi.2010, 11.i.2011, 6.ii.2011, 16.x.2011, 6.xi.2011, 3.vi.2012, ; Horshat Tal Nature Reserve, 30.v.2010, 14.ii.2012, 27.ii.2011, 13.iii. 2012, ; Alonei Abba Nature Reserve, 11.1.2011, (ICVI MC:385). First-instar nymphs; Yehudiya Nature Reserve, 6.iii.2010, 20.iii.2011, 24.iii.2012, ; Horshat Tal Nature Reserve, 13.iii.2012, 15.iii.2012, .Comparative material examinedNidularia pulvinata: France: adult female and first-instar nymphs, Serignan, Vauctuse, on Quercus ilex, 18.v.1978, I. Foldi (ICVI C:4946); first-instar nymphs, Caumont (Avignon), on Quercus ilex, 11.iv.1978, D. Matile, J.P. Fabre, (MNHN 7337-4); Italy: adult femalePisa, on Quercus ilex, 5.iv.1988, D. Matile-Ferrero Kermes quercus: Sweden: adult female, Skan, Near Lund on Quercus robur, 10.vi.2010, C.A Gertsson (ICVI C:4806); England: adult female , Wytham Wood, Berkshire on Quercus robur, 10.v.1965, S.C. Varley (BMNH 81-539); Poland; first-instar nymph, Warsaw on Quercus robur, 25.viii.1994, E. Podsiadlo (ICVI C:4798)Kermes roboris: Hungary: adult female, Budapest, Plant Protection Institute-adjacent track on Quercus sp., 8.vi.1989, C.P. Malumphy (BMNH); England: adult female, Kent: Herne Bay on Quercus spp., 00.viii.1899, C.D Waterhouse (ICIV C:5071).Kermes nahalali Bodenheimer, Kermes echinatus Balachowsky, Kermes greeni Bodenheimer, Kermes quercus (Linnaeus), Kermes spatulatus Balachowskyand Nidularia balachowskii. Three specimens of each species were used as replicates except for Nidularia balachowskii, where six were used. Adult females, preserved in 96% ethanol, were examined under the stereomicroscope for the presence of hymenopteran parasitoid wasps prior to DNA extraction. Voucher specimens were slide-mounted using the cuticle of the actual specimens from which DNA was extracted. Slide mounting followed the protocol outlined in Ben-Dov, Coccoidea: Asterolecaniidae, Coccidae, Diaspididae, Eriococcidae, Monophlebidae and Pseudococcidae. A species of aphid, Acyrthosiphon pisum , was used as the outgroup species. These sequences were made available from GenBank  method . PolymerPageBreakPageBreakPageBreak by 40 cycles of 95°C for 1 min, 45°C for 1 min, and 72°C for 1.5 min, with a final extension at 72°C for 5 min. Each reaction was examined by electrophoresis and bands were visualized with UV light. PCR products were excised from the gel and purified using the Zymoclean Gel Extraction Kit . Purified PCR products were sequenced in both the forward and reverse directions at Hy-Labs .The PCR cycling conditions for 28S were 94°C for 4 min, followed by 35 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1.5 min, with a final extension at 72°C for 4 min. The PCR cycling protocol for COI was 95°C for 7 min, followedSequence alignments for both 28S and COI gene sequences were performed with MUSCLE 3.7  and the Bodenheimerhttp://species-id.net/wiki/Nidularia_balachowskiiNidularia balachowskii Bodenheimer, 1941: 78–80.General appearance.Young, pre-reproductive adult dorsum brownish and venter yellowish white; oval, soft and flat; 1.2–1.9 mm long and 0.6–0.9 mm wide. Dorsal surface covered with 5 longitudinal rows of rectangular wax plates, each plate about 0.25 mm long and 0.3 mm wide; median row with 11 plates, lateral row on each side of median row with 9–11 plates and marginal rows with 7–9 plates. The wax plates become gradually smaller in size towards anterior and posterior apices and lateral margin : coxae 25–30, trochanter + femur 63–80, tibia 25–38, tarsus 25–60, claw 13–23; total leg length 158–213; trochanter with 2 oval, sensory pores on each side, each about 2–3 µm wide; setae present on each leg segment; tarsal digitules each 25–30 µm long, knobbed apically, extending beyond apex of claw; claw digitules knobbed apically, each 14–20 µm long, shorter than tarsal digitules; each claw with a single denticle near the tip. Clypeolabral shield well-developed; 75–90 µm long and 63–75 µm wide. Labium 3-segmented, triangular, 82–100 µm long and 35–38 µm wide; labial setae as follows; basal segment with 2 pairs of setae, each 10–15 µm long, median segment with 1 pair of hair-like setae on dorsal surface, each 10–20 µm long, apical segment with 3 pairs of hair-like setae, 12–17 µm long. Spiracles subequal in size; peritreme about 3–5 µm diameter; crescent shaped sclerosis 15–25 µm long. Bilocular pores, oval, each 2 µm long and 1 µm wide, with 1 present submarginally about level of each spiracle. Quinquelocular pores each 3 µm diameter, as follows; 1 just anterior to each spiracle; 1 medially to each coxa; 2 on each of abdominal segments IV, V, VI. Microspines present on median and submedian areas of each abdominal segment, in 2–4 transverse rows; each spine about 1 µm long. Setae 1 pair, similar in size and shape to marginal setae, between anterior apex of body and basal segments of antennae; 6 interantennal setae, each 20–30 µm long between basal segments of antennae and anterior apex of clypeus; 1 seta 8–15 µm long medially to each coxa; 2 longitudinal rows, each with 7–8 setae, similar in shape to marginal setae but shorter, each about 8 µm long, extending submarginally from laterad to metathoracic coxae to anterior margin of anal ring, no setae present on most posterior abdominal segment; 2 longitudinal rows of 6 setae, each about 5 µm long, located submarginally on abdomen; 2 longitudinal rows of 4 setae, each about 10 µm long, located submedially on abdomen; plus 2 longitudinal rows of 6 setae, about 17–18 µm long, located medially. Anal ring located ventrally; composed of 2 semi-circles; diameter 15–25 µm; each semi-circle with about 17 cells and 3 pointed setae, subequal in size, each 20–30 µm long. Also with a pair of setae, each 12–15 µm long, anterior to anal ring and 1 pair, each 15–25 µm long, postero-laterally to anal ring. Anal lobes well-developed; each lobe bearing 2 pointed setae, 12–15 µm long, distinctly thicker than marginal setae, plus 1 pair of long, apical setae 77–125 µm long.PageBreakMolecular identification and relationshipsMolecular resultsNidularia balachowskii  and from five adult female Palaearctic Kermesidae species . 28S gene sequences (~700 bp) and COI sequences (~400 bp) from all species were recovered and aligned with sequences of Coccoidea species representing different families (obtained from GenBank). All species for which multiple specimens were sampled showed no interspecies variation. The maximum likelihood analysis of both genes resulted in tree typologies that show that Nidularia balachowskii is a distinct species within the monophyletic Kermesidae. Nidularia balachowskii is grouped together with other kermesid species and not with the other Coccoidea  whitish eggs. Each egg was about 0.4 mm long and 0.2 mm wide. Once all of the eggs have been laid and the brood chamber full of eggs, the female dies and the dorsum becomes sclerotized. The sclerotized, convex body of the dead, post-reproductive female may remain on the host tree for a year or more after first-instar emergence.PageBreakruary. The females continue feeding and increase in body size throughout this period. Feeding was confirmed by observations of honeydew elimination. By late February, the dorsum of the female begins to expand greatly, increasing in convexity and sclerotization. The ventral surface of the abdomen becomes concave, forming the brood chamber into which the eggs are deposited. The ovipositing female secretes a woolly, white wax that surrounds its body margin. No injury has been observed to the oak hosts by Nidularia balachowskii in Israel.Eclosion of first-instar nymphs occurs inside the brood chamber and nymphs emerge from the cavity under the dead female body. This takes place from end of March and throughout April. Crawlers settle in bark crevices on branches and on the trunks of the trees. Young teneral females are found on the branches from June to FebNidularia balachowskii in Israel to Quercus ithaburensis trees, whereas Bodenheimer gives Quercus sp. as the host tree in Turkey  in PageBreak the spiracles, and tubular ducts on the venter.These characters are some of the synapomorphic characters of kermesid adult females that have been described by Some morphological characters of adult female Nidularia, adult female Nidularia balachowskii share with Nidularia pulvinata the following characters: (i) one-segmented antennae; (ii) absence of legs; (iii) absence of setae-pore clusters on venter; (iv) ventral position of anal ring, and (v) an anal ring with setae and cells, whereas the two Kermes species have: four, five or six-segmented antennae, and possess legs, setae-pore clusters on venter, anal ring placed on dorsum, and an anal ring without setae and cells. Comparing Nidularia balachowskii and Nidularia pulvinata, the most obvious distinguishing feature is the presence of quinquelocular pores on the spiracle peritreme of Nidularia pulvinata.Within the genus The morphological characters of first-instar nymphs of Nearctic kermesids were summarized by Nidularia balachowskii share the following characters with other Kermesidae species: (i) six-segmented antennae; (ii) three-segmented labium; (iii) simple pores forming longitudinal lines on the dorsum (iv) dorsal setae; (v) anal PageBreakring with cells and setae; (vi) microspines in rows on abdominal segments, and (vii) bilocular pores on venter. This last character is sometimes overlooked.The first-instar nymphs of Nidularia balachowskii can be distinguished from other Kermesidae species by the form of its marginal setae. Nidularia balachowskii has sharply spinose, apically pointed and slightly curved setae, each 9–15 µm long. This differs from Nidularia pulvinata which has hair-like setae (Nidularia japonica which has setose setae that are somewhat conical at the base (Kermes species that have been described possess conical, hair-like or spatulate marginal setae (Nidularia from the Palaearctic Kermes examined.ke setae  and Niduthe base .The firsal setae . There iNidularia balachowskii, six other species of Palaearctic Kermesidae, and species representing six other Coccoidea families showed that gene fragments of both COI and 28S separated Nidularia balachowskii from other Coccoidea species, and clearly placed Nidularia balachowskii in the Kermesidae. This study confirms that Nidularia balachowskii is a distinct species, clearly distinguishable from other closely-related kermesid species.The DNA-sequence data for Nidularia balachowskii, the identity of Nidularia balachowskii and its placement in the Kermesidae has been substantiated, and we have shown that Nidularia balachowskii is congeneric with Nidularia pulvinata.Based on both the morphological and molecular studies of


There were errors in the Funding section. The correct funding information is as follows: FL-D and AM were funded by National Institute of Health grant R01KD064842-07A1. QD was funded by National Institute of Health grant GM079506. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


HLA abnormalities on tumour cells for immune escape have been widely described. In addition, cellular components of the tumour microenvironment, in particular myeloid derived suppressor cells (MDSC) and alternatively activated M2 tumour-associated macrophages (TAMs), are involved in tumour promotion, progression, angiogenesis and suppression of anti-tumour immunity. However, the role of HLA in these activities is poorly understood. This review details MHC class I characteristics and describes MHC class I receptors functions. This analysis established the basis for a reflection about the crosstalk among the tumour cells, the TAMs and the cells mediating an immune response.The tumour cells and TAMs exploit MHC class I molecules to modulate the surrounding immune cells. HLA A, B, C and G molecules down-regulate the macrophage myeloid activation through the interaction with the inhibitory LILRB receptors. HLA A, B, C are able to engage inhibitory KIR receptors negatively regulating the Natural Killer and cytotoxic T lymphocytes function while HLA-G induces the secretion of pro-angiogenic cytokines and chemokine thanks to an activator KIR receptor expressed by a minority of peripheral NK cells. The open conformer of classical MHC-I is able to interact with LILRA receptors described as being associated to the Th2-type cytokine response, triggering a condition for the M2 like TAM polarization. In addition, HLA-E antigens on the surface of the TAMs bind the inhibitory receptor CD94/NKG2A expressed by a subset of NK cells and activated cytotoxic T lymphocytes protecting from the cytolysis.Furthermore MHC class II expression by antigen presenting cells is finely regulated by factors provided with immunological capacities. Tumour-associated macrophages show an epigenetically controlled down-regulation of the MHC class II expression induced by the decoy receptor DcR3, a member of the TNFR, which further enhances the M2-like polarization. BAT3, a positive regulator of MHC class II expression in normal macrophages, seems to be secreted by TAMs, consequently lacking its intracellular function, it looks like acting as an immunosuppressive factor.In conclusion HLA could cover a considerable role in tumour-development orchestrated by tumour-associated macrophages. Macrophages are tissue phagocytic cells that display the main activity of clearance independently of immune cells. However, they can be activated in response to either innate or adaptive immune signals and they are able to shape their phenotype to fit the microenvironment. In an attempt to simplify their complexity, activated macrophages can be categorized into two distinct subtypes: classically activated M1) and alternatively activated M2)  Growth Factor; HIV: Human Immunodeficiency Virus; HLA: Human Leucocyte Antigen; ICOS: Inducible T-cell Costimulator; IL: Interleukin; ILT: Immunoglobulin-Like Transcript 2; INF: Interferon; iNOS: inducible NO Synthase; KIR: Killer cell Immunoglobulin-like Receptor; LILR: Leukocyte Immunoglobulin-like Receptor; LPS: Lipopolysaccarhide; M: Macrophage; MHC: Major Histocompatibility Complex; NSLC: Non-Small Lung Cancer; mRNA: messenger RiboNucleoAcid; MSC: Mesenchymal Stem cell; MDSC: Myeloid-derived Suppressor Cell; NHL: Non-Hodgkin Lymphoma; NK: Natural Killer; Stat: Signal transducer and activator of transcription protein; TAP: Transporter associated with antigen processing; TAM: Tumour-Associated Macrophage; TCR: T-Cell Receptor; TGF: Tumour Growth Factor; TNFR: Tumour Necrosis Factor Receptor; VEGF: Vascular Endothelial Growth Factor.The authors declare that they have no competing interests.GVM is the project leader and he has designed the review, contributed with suggestions to the editing; MM has contributed to literature research and writing and concepts development; EA, LV, BS, AL and RK have contributed with specific editing and suggestion that regards their specific area of interest. All authors agreed and contributed to conceived the study, and participated to the final draft the manuscript. All authors read and approved the final manuscript.