Microvascular Hyperplasia Research Articles

Comparing qSMI and qCEUS for assessing vascularization in uterine cervical cancer: operable versus non-operable group.

The present study aimed to compare the effectiveness and reliability of quantified superb microvascular imaging (qSMI) and quantified contrast-enhanced ultrasonography (qCEUS) in assessing vascularization in both operable and non-operable uterine cervical cancer. A case-control study included 64 patients with pathology-proven and untreated cervical cancer, who underwent transvaginal ultrasonography combined with qSMI and qCEUS between January 2022 and June 2023. SMI results were quantified as the vascular index (VI), which were compared to 12 quantitative parameters of CEUS calculated with time-intensity curves (TIC). According to FIGO staging and different treatment regimens, 64 patients with cervical cancer were divided into operable group (IA ~ IIA, n = 19) and non-operable group (IIB ~ IV, n = 45). In comparison to the operable group, the non-operable group showed significantly higher values in VI, peak intensity (PI), area under the curve (AUC), wash-in area (iAUC), wash-out area (oAUC), wash-in rate (WiR), mean intensity (Mean Int), and standard deviation (STD) (all P < 0.05). VI demonstrated strong correlations with CEUS parameters, notably PI (r = 0.854, P < 0.001) and AUC (r = 0.635, P < 0.001). Furthermore, VI showed a better predictive performance for treatment-group assignment than qCEUS parameters, with an 80.7% accuracy, 64.4% sensitivity and 89.5% specificity. Both qSMI and qCEUS exhibit significant and comparable utility in detecting microvascular hyperplasia and predicting treatment-group assignments in cervical cancer. Furthermore, qSMI may offer added convenience in implementation.

CS-10 POLYMORPHOUS LOW-GRADE NEUROEPITHELIAL TUMOR OF THE YOUNG: OUR CASE AND REVIEW OF THE LITERATURE

Abstract Polymorphous low-grade neuroepithelial tumor of the young (PLNTY) is a new tumor that was officially listed in the WHO 5th edition in 2021. It is clinically and pathologically oligodendroglioma-like, but can be distinguished by CD34 positivity. Although this case was initially thought to be an oligodendroglioma-like low grade glioma, it was clinically and pathologically considered to be a typical case, so we report it here.14 year old female. No special notes, including growth and development. The patient suddenly developed a generalized tonic convulsion and was taken to the emergency room. A CT scan revealed a lesion with strong calcification in her left temporal lobe, and she was referred to our department. She had no neurological findings and underwent total resection under awake surgery. She was difficult to diagnose with intraoperative frozen section. diagnosis.The pathological findings are medium-sized oval to oblong nuclei, diffuse proliferation of tumor cells with delicate cell projections, and oligodendroglioma-like areas with perinuclear halos. A very large number of fine calcifications are observed, but necrosis and microvascular hyperplasia are not observed. GFAP, olig2, and nestin were positive, and synaptophysin, Nf, Hu C/D, MAP2, and chromograninA were negative. MIB-1 was less than 5%. The sequence was IDH1/2, TERTwild, and the BRAF-V600E mutation was recognized. Tumor cells were CD34-positive, leading to a final diagnosis of PLNTY. PLNTY is a new concept of tumor and accumulation is still small. Although this case has typical clinical and pathological features, preoperative diagnosis was difficult because it is a rare tumor. Intraoperative diagnosis is also difficult, suggesting the difficulty of diagnosing this tumor. Based on past cases, we report preoperative diagnostic imaging, intraoperative findings, and histopathological findings, including literature considerations.

β-Catenin marks proliferating endothelial cells in glioblastoma

BackgroundAngiogenesis is a key process in the growth and maintenance of tumors. The Wnt signaling pathway is required for angiogenesis of the central nervous system though development of the blood–brain barrier and subsequent proliferation of endothelial cells during tumor growth. However, the specificity of the Wnt pathway in regulating endothelial cells of different central nervous systems remains to be investigated. Materials & methodsPatient-derived tissue samples from 35 paraffin-embedded tumors were used to assess β-catenin immunoexpression. Tumor samples consisted of the following pathologies: grade II diffuse astrocytoma, glioblastoma, hemangioblastoma, and metastatic adenocarcinoma (lung or breast primary). Average percent reactivity was recorded as a mean observed in ten high-power fields. The following scale was used to grade immunoreactivity: 0 = immunonegative, 1 = 1–25% reactive, 2 = 26–50% reactive, 3 = 51–75% reactive, 4 = 76–100% reactive. ResultsWhile we did not observe nuclear expression of β-catenin in any samples, there was uniform cytoplasmic expression of β-catenin within glial tumor cells. There was a clear distinction in tumor endothelial cells whereby diffuse staining was noted in areas of microvascular hyperplasia in GBM and a less immunoreactive profile in low-grade astrocytomas. By contrast, non-glial tumors, contained very minimal cytoplasmic β-catenin expression in tumor and stromal cells and were devoid of immunoreactivity in endothelial cells. Conclusionβ-catenin is unique marker of proliferating endothelial cells in GBM. Therapies targeting the spatial and structural heterogeneity inherent to GBM may prove to be efficacious and result in an improved survivorship.

Clear cell renal cell carcinoma with prominent microvascular hyperplasia: Morphologic, immunohistochemical and molecular-genetic analysis of 7 sporadic cases

Clear cell renal cell carcinoma (CCRCC) is well known for intratumor heterogeneity. An accurate mapping of the tumor is crucial for assessing prognosis, and perhaps this can be linked to potential success/failure of targeted therapies.We assembled a cohort of 7 CCRCCs with prominent vasculature and microvascular hyperplasia (ccRCCPV), resembling those seen in high grade gliomas. A control group of classic CCRCC with no variant morphologies was also included. Both groups were analyzed for clinicopathologic, morphologic, immunohistochemical, and molecular genetic features.No statistically significant differences in mRNA expression of studied genes between the two groups were found. Using NGS panel Trusight Oncology 500 (TSO500), only one clinically significant gene mutation, VHL c.263G > A, p. (Trp88Ter), was found. TMB (Tumor Mutation Burden) and MSI (MicroSatellite Instability) were low, and no copy number variations (CNVs) were detected in the study cohort.Prominent microvascular hyperplasia in CCRCC is a rare phenomenon. From molecular genetic point of view, these tumors do not appear to be different from classic CCRCC. Prognostically, they also demonstrated similar clinical behaviors.

Sulforaphane Causes Cell Cycle Arrest and Apoptosis in Human Glioblastoma U87MG and U373MG Cell Lines under Hypoxic Conditions.

Glioblastoma multiforme (GBM) is the most prevalent and aggressive primary brain tumor. The median survival rate from diagnosis ranges from 15 to 17 months because the tumor is resistant to most therapeutic strategies. GBM exhibits microvascular hyperplasia and pronounced necrosis triggered by hypoxia. Sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, has already demonstrated the ability to inhibit cell proliferation, by provoking cell cycle arrest, and leading to apoptosis in many cell lines. In this study, we investigated the antineoplastic effects of SFN [20–80 µM for 48 h] in GBM cells under normoxic and hypoxic conditions. Cell viability assays, flow cytometry, and Western blot results revealed that SFN could induce apoptosis of GBM cells in a dose-dependent manner, under both conditions. In particular, SFN significantly induced caspase 3/7 activation and DNA fragmentation. Moreover, our results demonstrated that SFN suppressed GBM cells proliferation by arresting the cell cycle at the S-phase, also under hypoxic condition, and that these effects may be due in part to its ability to induce oxidative stress by reducing glutathione levels and to increase the phosphorylation of extracellular signal-regulated kinases (ERKs). Overall, we hypothesized that SFN treatment might serve as a potential therapeutic strategy, alone or in combination, against GBM.

A prospective study of the effect and mechanism of autologous platelet-rich plasma combined with Meek microskin grafts in repairing the wounds of limbs in severely burned patients

Objective: To observe the effect of autologous platelet-rich plasma (PRP) combined with Meek microskin grafts in repairing the wounds of limbs in severely burned patients, and to explore the mechanism. Methods: The prospective controlled research method was used. From September 2016 to January 2020, 16 patients aged 18-69 years, with extensive deep burns, including 9 males and 7 females, who met the selection criteria were admitted to the Department of Burns and Plastic Surgery of the 909th Hospital of the Joint Logistic Support Force of PLA. The bilateral limbs with similar injury in 8 patients were divided into Meek skin grafting+PRP group and Meek skin grafting alone group according to the random number table; in the other 8 patients, the limbs with severer injury were included in Meek skin grafting+PRP group, and the limbs on the other side were included in Meek skin grafting alone group. The wounds of affected limbs in the two groups were treated correspondingly. On post surgery day (PSD) 10, the survival and fusion of Meek microskin grafts were observed and the survival rate and fusion rate were calculated; the histological morphology and the angiogenesis of the basal tissue of Meek microskin graft were observed by hematoxylin-eosin staining and immunohistochemical staining, respectively, with the microvessels being counted. Data were statistically analyzed with paired sample t test. Results: On PSD 10, the wounds of affected limbs in Meek skin grafting+PRP group were dry, and most of the transplanted skin grafts were closely adhered to the basal tissue; while a small amount of exudate could be found in the wounds of affected limbs in Meek skin grafting alone group, and a small part of the transplanted microskin grafts fell off or poorly attached to the basal tissue. On PSD 10, the survival rate and the fusion rate of Meek microskin grafts in the wounds of affected limbs in Meek skin grafting+PRP group were (94±3)% and (86±4)%, which were significantly higher than (89±4)% and (79±4)% of Meek skin grafting alone group, respectively (t=3.633, 4.229, P<0.01). On PSD 10, the basal epidermis was closely connected with dermis of Meek microskin grafts in the wounds of affected limbs in Meek skin grafting+PRP group, with more inflammatory cell infiltration and active microvascular hyperplasia, while the basal epidermis was less closely connected with dermis of Meek microskin grafts in the wounds of affected limbs in Meek skin grafting alone group, with obvious degeneration of collagen fibers under the dermis, less inflammatory cell infiltration, and slightly poor microvascular hyperplasia. On PSD 10, the distribution of microvessels in basal tissue of Meek microskin grafts in the wounds of affected limbs in Meek skin grafting+PRP group were densely clustered, while the distribution of microvessels in Meek skin grafting alone group were scattered, sparse, and dotted. On PSD 10, the number of microvessels in basal tissue of Meek microskin grafts in the wounds of affected limbs in Meek skin grafting+PRP group was 36±6 in each 400-fold visual field, which was significantly more than 29±7 of Meek skin grafting alone group (t=2.671, P<0.05). Conclusions: Autologous PRP can effectively promote the survival rate and fusion rate of Meek microskin grafts in the wounds of limbs after escharectomy in severely burned patients by promoting angiogenesis at the base of Meek microskin grafts.

This Is a Title in Title Case: SDF1,CXCR4 and MVD Play Important Roles in Multiple Myeloma

The myeloid microenvironment of patients with multiple myeloma plays an important role in regulating the occurrence, development, invasion, secretion and angiogenesis of myeloma cells. Chemokines SDF-1 and its receptor CXCR4 form SDF1-CXCR4 biologicalaxis in bone marrow microenvironment. The role and mechanism of SDF1-CXCR4 biologicalaxis in multiple myeloma remain to be studied.We collected 144 cases of hematology bone marrow from January 2013 to June 2017 in Southwest Medical University Affiliated Hospital and the Third People's Hospital of Chengdu, of which 104 cases were multiple myeloma patients, and 40 cases were normal as control. The relationship between SDF1-CXCR4 expression and its clinical features were analyzed. Through these analysis we tried to learn the role of SDF1-CXCR4 in the development, progression and prognosis of multiple myeloma, and hoped to provide evidence for the diagnosis and treatment of multiple myeloma.As a result, the positive expression of SDF1 and CXCR4 in bone marrow biopsy of patients with multiple myeloma was significantly higher than that in the normal control group. More importantly, there was a positive correlation between the expression of SDF1 and CXCR4, indicating that SDF1-CXCR4 in bone marrow microenvironment plays an important role in the occurrence of multiple myeloma.The density of microvessel density (MVD) in the myeloma was also significantly higher than that in the normal control group. The expression of SDF1-CXCR4 in the myeloma group was positively correlated with the expression of MVD. This study demonstrates that multiple myeloma cell growth and microvascular proliferation are significantly correlated in microenvironment. It is suggested that the secretion of chemokine SDF1-CXCR4 in the bone marrow microenvironment of multiple myeloma may promote microvascular proliferation and the growth of tumor cells, which may be the pathogenesis.Multiple myeloma clinical features were analyzed according to SDF1-CXCR4 and MVD expression levels. We found that SDF1-CXCR4 and MVD expression levels were not correlated with gender, age and immunoglobulin typing, but were significantly correlated with multiple myeloma staging in bone marrow tissue of MM patients. But we found that in the later stage of SDF1, the expression level of SDF1、CXCR4 and MVD is higher. This finding further indicates that chemokine SDF1-CXCR4 secretion and microvascular proliferation play important roles in progression of multiple myeloma.The Kplan-Meier analysis on overall survival (OS) and progression-free survival (PFS) was performed according to the levels of SDF1-CXCR4 and MVD expression in multiple myeloma. We discovered that the OS rate and PFS rate were lower in the patients with higher expression of SDF1, CXCR4 and MVD , which indicates that chemokine SDF1-CXCR4 secretion and microvascular proliferation also play important roles in prognosis of multiple myeloma.Our findings reveal that chemokine SDF1-CXCR4 secretion and microvascular hyperplasia in bone marrow microenvironment are essential in the occurrence, development and prognosis of multiple myeloma. The higher secretion of chemokine SDF1-CXCR4 suggests the worse progression and poor prognosis. Chemokine SDF1-CXCR4 is expected to become a new target for MM treatment. DisclosuresNo relevant conflicts of interest to declare.

Multiscale modeling of glioma pseudopalisades: contributions from the tumor microenvironment

Gliomas are primary brain tumors with a high invasive potential and infiltrative spread. Among them, glioblastoma multiforme (GBM) exhibits microvascular hyperplasia and pronounced necrosis triggered by hypoxia. Histological samples showing garland-like hypercellular structures (so-called pseudopalisades) centered around the occlusion site of a capillary are typical for GBM and hint on poor prognosis of patient survival. We propose a multiscale modeling approach in the kinetic theory of active particles framework and deduce by an upscaling process a reaction-diffusion model with repellent pH-taxis. We prove existence of a unique global bounded classical solution for a version of the obtained macroscopic system and investigate the asymptotic behavior of the solution. Moreover, we study two different types of scaling and compare the behavior of the obtained macroscopic PDEs by way of simulations. These show that patterns (not necessarily of Turing type), including pseudopalisades, can be formed for some parameter ranges, in accordance with the tumor grade. This is true when the PDEs are obtained via parabolic scaling (undirected tissue), while no such patterns are observed for the PDEs arising by a hyperbolic limit (directed tissue). This suggests that brain tissue might be undirected - at least as far as glioma migration is concerned. We also investigate two different ways of including cell level descriptions of response to hypoxia and the way they are related .

Clinicopathological, Immunohistochemical and Molecular Genetic Study on Epithelioid Glioblastoma: A Series of Fifteen Cases with Literature Review.

PurposeTo observe the clinicopathological, immunohistochemical, and molecular genetic features of epithelioid glioblastoma (E-GBM), and identify tumor-associated prognostic factors.Patients and MethodsThe clinical and radiological data of fifteen cases of E-GBM were collected, and their pathological, immunohistochemical, and molecular features were examined. A 1p/19q analysis via FISH, MGMT promoter methylation by MS-PCR, and IDH1 and BRAF V600E mutation analysis by HRM-PCR were performed. The level of EZH2 expression was valuated by immunohistochemistry in 15 E-GBM cases, and the prognostic factors were analyzed in E-GBM patients. Fifteen non-E-GBM cases were used as a control.ResultsThe fifteen cases of E-GBM included twelve males and three females, with fourteen cases supratentorially located. Headache was the main symptom. Microscopy revealed that the tumors were composed of epithelioid cells and some rhabdoid cells. The epithelioid and rhabdoid cells displayed focal discohesion, scant intervening neuropil, a distinct cell membrane, eosinophilic cytoplasm, and a laterally positioned nucleus. Most tumors showed high mitosis, zonal necrosis, and microvascular hyperplasia. Immunohistochemical findings included epithelioid cells positive for GFAP, vimentin, nestin, S-100, and INI-1. The molecular findings included no deletions of 1p/19q, EGFR amplifications, or IDH1 mutations in any case, a methylated MGMT promoter in 46.7% (7/15) cases, and a BRAFV600E mutation in 46.7% (7/15) cases. EZH2 overexpression occurred in 60.0% (9/15) of E-GBM cases. E-GBM patients with OS (≤12 months) exhibited extensive necrosis (6/6), EZH2 overexpression (6/6), MGMT promoter unmethylation (5/6), BRAFV600E mutation (3/6), and treatment (surgery4/6). E-GBM patients with OS (>12 months) exhibited focal or limited necrosis, low or negative EZH2 expression, MGMT promoter methylation (2/3), BRAFV600E mutation (3/3), and treatment (surgery+radiotherapy/chemo-radiotherapy, 2/3).ConclusionE-GBM was a rare variant of glioblastoma, with histological epithelioid features and poor prognosis. Extensive necrosis, MGMT promoter unmethylation, EZH2 overexpression, and lack of adjuvant chemo-radiotherapy may indicate a poor prognosis.

MCT4 regulates de novo pyrimidine biosynthesis in GBM in a lactate-independent manner.

BackgroundNecrotic foci with surrounding hypoxic cellular pseudopalisades and microvascular hyperplasia are histological features found in glioblastoma (GBM). We have previously shown that monocarboxylate transporter 4 (MCT4) is highly expressed in necrotic/hypoxic regions in GBM and that increased levels of MCT4 are associated with worse clinical outcomes.MethodsA combined transcriptomics and metabolomics analysis was performed to study the effects of MCT4 depletion in hypoxic GBM neurospheres. Stable and inducible MCT4-depletion systems were used to evaluate the effects of and underlining mechanisms associated with MCT4 depletion in vitro and in vivo, alone and in combination with radiation.ResultsThis study establishes that conditional depletion of MCT4 profoundly impairs self-renewal and reduces the frequency and tumorigenicity of aggressive, therapy-resistant, glioblastoma stem cells. Mechanistically, we observed that MCT4 depletion induces anaplerotic glutaminolysis and abrogates de novo pyrimidine biosynthesis. The latter results in a dramatic increase in DNA damage and apoptotic cell death, phenotypes that were readily rescued by pyrimidine nucleosides supplementation. Consequently, we found that MCT4 depletion promoted a significant prolongation of survival of animals bearing established orthotopic xenografts, an effect that was extended by adjuvant treatment with focused radiation.ConclusionsOur findings establish a novel role for MCT4 as a critical regulator of cellular deoxyribonucleotide levels and provide a new therapeutic direction related to MCT4 depletion in GBM.

TMOD-35. PREDICTION OF OVERALL SURVIVAL, AND MOLECULAR MARKERS IN GLIOMAS VIA ANALYSIS OF DIGITAL PATHOLOGY IMAGES USING DEEP LEARNING

Abstract BACKGROUND Microscopic features of brain tumors, such as tumor cell morphology, type/degree of microvascular hyperplasia, mitotic activity, and extent of zonal/geographic necrosis, among many others, are measurable and reflect underlying molecular markers that are predictive of patient prognosis, signifying that quantitative analysis may provide insight into disease mechanics. We developed a computational method to predict overall-survival and molecular markers for brain tumors using deep learning on whole-slide digital images (WSDI). METHODS The WSDI were acquired from TCGA for 663 patients [IDH:333 wildtype, 330 mutants, 1p/19q:201 non-codeleted, 129 codeleted]. A set of 100 region-of-interest each comprising 1024x1024 that contained viable tumor with descriptive histologic characteristics and that were free of artifacts were extracted. A modified version of ResNet with architecture of 50 convolutional layers was used. The network was optimized using stochastic gradient decent optimization method with binary cross-entropy loss. Sigmoid- and linear-activation were, respectively, used as final layer for mutation and survival prediction. Data was divided into training (50%), testing (25%), and validation (25%). RESULTS The model predicted IDH and 1p/19q with an accuracy of 88.92%[sensitivity(se)/ specificity(sp)=87.77/84.35] and 88.23%(se/sp=87.38/88.58), respectively. The accuracy was further improved, when classification was done within homogeneous grades, for IDH [II=90.50%(se/sp=91.52/78.57), III=91.21%(se/sp=91.89/89.47), IV=92.77%(se/sp=77.78/93.51)] and 1p/19q [II=91.51%(se/sp=91.30/91.66), III=92.56(se/sp =93.33,92.04)]. The Pearson correlation coefficient between the predicted scores and overall-survival was 0.79 (p&lt; 0.0001). CONCLUSION Our findings suggest that deep learning techniques can be applied to WSDI for objective, and accurate prediction of mutations and survival. Our approach, when compared with expensive molecular based assays that invariably capture molecular markers from a small part of the tumor and also destroy the tissue, could (i) offer the same service at a reduced price, (ii) enable disease characterization across the entire landscape of the tissue, (iii) be beneficial for tissues inadequate for molecular testing, and (iv) does not need physical shipping of the tissue.

MiR145-5p inhibits proliferation of PMVECs via PAI-1 in experimental hepatopulmonary syndrome rat pulmonary microvascular hyperplasia.

ABSTRACTHepatopulmonary syndrome (HPS) is a triad of advanced liver disease, intrapulmonary vasodilatation and arterial hypoxemia. Increasing evidence shows that HPS is associated with pulmonary microvascular hyperplasia. The aim of this work was to investigate the underlying mechanism of miR-145 in regulating the proliferation of pulmonary microvascular endothelial cells (PMVECs) and angiogenesis in HPS via plasminogen activator inhibitor-1 (PAI-1). To test this, morphology score and number of pulmonary microvascular were assessed in lung tissues from rats with HPS by Hematoxylin and Eosin (H&E) staining. Expression levels of PAI-1 were assessed in lung tissues from HPS rats, as well as in PMVECs treated with HPS rat serum. We also selected the putative microRNA binding site on PAI-1 by bioinformatics analysis. Then, miR145-3p and miR145-5p expression levels in the lungs and PMVECs of rats were detected by qRT-PCR because miR145-5p is a microRNA binding site on PAI-1. In addition, the effects of miR-145-5p regulation on PAI-1 were examined by upregulation and downregulation of miR-145-5p and specific lentivirus transfection was used to overexpress and knockdown PAI-1 to assess PAI-1 function on PMVECs proliferation. Our data showed that levels of PAI-1 expression in lung tissue of rats increased significantly when rats were treated with common bile duct ligation. We found that levels of miR-145-5p were frequently downregulated in HPS tissues and cell lines, and overexpression of miR-145-5p dramatically inhibited PMVECs proliferation. We further verified PAI-1 as a novel and direct target of miR-145-5p in HPS. MiR-145-5p inhibits PAI-1 synthesis and the expression changes of PAI-1 directly affect the proliferation of PMVECs. We concluded that miR-145-5p negatively regulates PMVEC proliferation through PAI-1 expression. In addition, overexpression of miR-145-5p may prove beneficial as a therapeutic strategy for HPS treatment.

Characterisation of Lamp2-deficient rats for potential new animal model of Danon disease

Danon disease (DD) is caused by the absence or malfunction of lysosomal-associated membrane protein 2 (LAMP2). Although Lamp2-deficient mice and DD patients have similar characteristics, these mice have clear limitations and are clinically inconsistent. The aim of our paper is to outline the characteristics of Lamp2-deficient rats and to contrast this model with currently available DD mouse models. The baseline levels of some serum enzymes were elevated in Lamp2y/− rats along with hypercholesterolemia and hyperglycaemia at 8 weeks. Echocardiography showed that IVSd (1.500 ± 0.071 vs. 2.200 ± 1.147, P < 0.01) and LVPWd (1.575 ± 0.063 vs. 1.850 ± 0.029, P < 0.01) were significantly increased, and GCS (−13.20 ± 0.4814 vs. −6.954 ± 0.665) and GRS (21.42 ± 1.807 vs. 7.788 ± 1.140) were sharply decreased. Meanwhile, substantial myocyte disruption, hypertrophic muscle fibres, interstitial fibrosis and microvascular hyperplasia could be observed in the heart tissue. Lamp2y/− rats also displayed abnormal behaviours in the open field and fear conditioning tests. Notably, Lamp2y/− rats manifested other system dysfunctions, such as retinopathy, chronic kidney injury and sterility. Based on these results, Lamp2-deficient rats exhibited greater similarity to DD patients in terms of onset and multisystem lesions than did mouse models, and these rats could be used as a valuable animal model for DD.

Glioblastoma Stem Cells Microenvironment: The Paracrine Roles of the Niche in Drug and Radioresistance.

Among all solid tumors, the high-grade glioma appears to be the most vascularized one. In fact, “microvascular hyperplasia” is a hallmark of GBM. An altered vascular network determines irregular blood flow, so that tumor cells spread rapidly beyond the diffusion distance of oxygen in the tissue, with the consequent formation of hypoxic or anoxic areas, where the bulk of glioblastoma stem cells (GSCs) reside. The response to this event is the induction of angiogenesis, a process mediated by hypoxia inducible factors. However, this new capillary network is not efficient in maintaining a proper oxygen supply to the tumor mass, thereby causing an oxygen gradient within the neoplastic zone. This microenvironment helps GSCs to remain in a “quiescent” state preserving their potential to proliferate and differentiate, thus protecting them by the effects of chemo- and radiotherapy. Recent evidences suggest that responses of glioblastoma to standard therapies are determined by the microenvironment of the niche, where the GSCs reside, allowing a variety of mechanisms that contribute to the chemo- and radioresistance, by preserving GSCs. It is, therefore, crucial to investigate the components/factors of the niche in order to formulate new adjuvant therapies rendering more efficiently the gold standard therapies for this neoplasm.

AI-02 * HYPOXIA AS A MEASURE OF FUNCTIONAL STATUS OF GLIOBLASTOMA VASCULATURE

INTRODUCTION: Glioblastoma (GBMs) exhibit distinct histopathological features including microvascular hyperplasia and heterogenous hypoxia within the tumor microenvironment. Newly formed vessels are often leaky/dysfunctional with intravascular thrombosis and potentially contribute to the hypoxic/necrotic foci seen throughout GBMs. By analyzing orthotopic xenograft models of GBM, we have established the correlation between tumor blood vessel and oxygen diffusion gradient. Additionally, we have established the effect of anti-angiogenic therapies (AATx) such as VEGF-Trap on this correlation. METHOD: Patient derived GSCs and established GBM cell line (U87) with or without VEGF-Trap were injected orthotopically in forebrains of NOD/SCID mice. Following 3-4 weeks of cell implantation, the mice were sacrificed and brains were harvested and imbedded in paraffin blocks. The sections were then stained for CD31 (endothelial marker) and CAIX (hypoxia marker). Images were scanned and quantified; using ImageJ (http://imagej.nih.gov/ij/), measurements include distance from tumor blood vessels to nearest hypoxic foci, total percent hypoxia, and cell proliferation. RESULTS: In literature it has been shown that any functional micro-vessel in normal tissue allows oxygen diffusion up to 100 µm radius. Using this as a guideline, we have defined the functional state of microvessels in our orthoptic tumor. We found that approximately 13% of the tumor blood vessels were dysfunctional, as their distance from immediate hypoxic cells was less than 20 µm. Upon treatment with AATx, we observed significant decrease in numbers of functional tumor blood vessels and cell prolliferation with concomitant increase in hypoxic areas within the tumor. CONCLUSIONS: AATx results in a significant decrease in the number of functional blood vessels - as measured by their proximity to the nearest hypoxic cells - and cell proliferation. We have provided a quantitative measure - distinct histopathological features including hypoxic and necrotic foci, microvascular hyperplasia and increased cell proliferation - for what was previously describe in qualitive terms in literature.

AI-11 * c-MET-MEDIATED ENDOTHELIAL-MESENCHYMAL TRANSITION DRIVES GLIOBLASTOMA PROGRESSION AND THERAPEUTIC RESISTANCE

Excessive and abnormal vasculature characterizes the microenvironment that fuels the progression of malignant tumors including glioblastoma multiforme (GBM), the grade IV glioma. GBM is among the most lethal human malignancies, distinguished by extensive microvascular hyperplasia due to intravessel endothelial cell (EC) proliferation with unknown etiology. Here, we show that c-Met phosphorylation induces endothelial-mesenchymal transition (Endo-MT), contributing significantly to vascular abnormality, GBM progression, and therapeutic resistance. Utilizing murine orthotropic GBM models induced by transplantation of GL26 glioma cells and RCAS/tv-a-mediated somatic gene transfer of platelet-derived growth factor (PDGF) in Ink-4a/Arf−/-PTEN−/- mice, we reveal robust Endo-MT in tumor vasculature, characterized by expression of the fibroblast-specific marker FSP-1 in 60% of CD31+ or CD105+ ECs, and the co-expression level correlates with the aggressiveness of glioma in humans. Cell fate analysis by lineage tracing Tie2+ ECs shows over half of FSP-1+ fibroblasts are of EC origin in the mouse GBM models. Tumor-conditioned medium induces in vitro EC acquisition of mesenchymal gene signature including FSP-1 and N-cadherin expression, confirming the microenvironment-dependent Endo-MT. Furthermore, proteomic analysis identifies a critical role of c-Met in Endo-MT, which is required for the increased EC proliferation and migration and vessel hyperpermeability in the GBM microenvironment. EC-specific c-Met phosphorylation induces MMP-14 expression, leading to Endo-MT and vascular abnormality. Finally, pharmacological inhibition of c-Met phosphorylation normalizes blood vessels, reduces vascular density and Endo-MT, blocks tumor progression, and sensitizes tumor to temozolomide treatment; all of which improves overall survival in the GBM-bearing mice. These findings reveal a novel mechanism controlling aberrant vascularization and GBM progression, and suggest that targeting c-Met phosphorylation and Endo-MT may offer selective and efficient therapeutic strategies for the treatment-resistant GBM, and possibly other malignant tumors.

Combined therapies of antithrombotics and antioxidants delayin silicobrain tumour progression

Glioblastoma multiforme (GBM), the most frequent type of primary brain tumour, is a rapidly evolving and spatially heterogeneous high-grade astrocytoma that presents areas of necrosis, hypercellularity and microvascular hyperplasia. The aberrant vasculature leads to hypoxic areas and results in an increase in oxidative stress, selecting for more invasive tumour cell phenotypes. In our study, we assay in silico different therapeutic approaches which combine antithrombotics (ATs), antioxidants and standard radiotherapy (RT). To do so, we have developed a biocomputational model of GBM that incorporates the spatio-temporal interplay among two glioma cell phenotypes corresponding to oxygenated and hypoxic cells, a necrotic core and the local vasculature whose response evolves with tumour progression. Our numerical simulations predict that suitable combinations of ATs and antioxidants may diminish, in a synergistic way, oxidative stress and the subsequent hypoxic response. This novel therapeutical strategy, with potentially low or no toxicity, might reduce tumour invasion and further sensitize GBM to conventional RT or other cytotoxic agents, hopefully increasing median patient overall survival time.

Super-Resolution Track Density Imaging of Glioblastoma: Histopathologic Correlation

Super-resolution track density imaging generates anatomic images with submillimeter voxel resolution by using high-angular-resolution diffusion imaging and fiber-tractography. TDI within the diseased human brain has not been previously described. The purpose of this study was to correlate TDI with histopathologic features of GBM. A total of 43 tumor specimens (24 contrast-enhancing, 12 NE, and 7 centrally necrotic regions) were collected from 18 patients with treatment-naïve GBM by use of MR imaging-guided neurosurgical techniques. Immunohistochemical stains were used to evaluate the following histopathologic features: hypoxia, architectural disruption, microvascular hyperplasia, and cellular proliferation. We reconstructed track density maps at a 0.25-mm isotropic spatial resolution by using probabilistic streamline tractography combined with constrained spheric deconvolution (model order, 8; 0.1-mm step size; 1 million seed points). Track density values were obtained from each tissue site. A P value of .05 was considered significant and was adjusted for multiple comparisons by use of the false discovery rate method. Track density was not significantly different between contrast-enhancing and NE regions but was more likely to be elevated within regions demonstrating aggressive histopathologic features (P < .05). Significant correlation between relative track density and hypoxia (odds ratio, 3.52; P = .01), architectural disruption (odds ratio, 3.49; P = .03), and cellular proliferation (odds ratio, 1.70; P = .05) was observed irrespective of the presence or absence of contrast enhancement. Numeric values of track density correlate with GBM biologic features and may be clinically useful for identification of regions of tumor infiltration within both enhancing and NE components of GBM.

Comparison of DSC‐MRI post‐processing techniques in predicting microvascular histopathology in patients newly diagnosed with GBM

To evaluate which common post-processing method applied to gradient-echo DSC-MRI data, acquired with a single gadolinium injection and low flip-angle, most accurately reflects microvascular histopathology for patients with de novo, treatment-naive glioblastoma multiforme (GBM). Seventy-two tissue samples were collected from 35 patients with treatment-naive GBM. Sample locations were co-registered to preoperative gradient-echo dynamic susceptibility contrast (DSC) MRI acquired with 35° flip-angle and 0.1 mmol/kg gadolinium. Estimates of blood volume and leakiness at each sample location were calculated using four common postprocessing methods (leakage-corrected nonlinear gamma-variate, non-parametric, scaled MR-signal, and unscaled MR-signal). Tissue sample microvascular morphology was characterized using Factor VIII immunohistochemical analysis. A random-effects regression model, adjusted for repeated measures and contrast-enhancement (CE), identified whether MR parameter estimates significantly predicted IHC findings. Elevated blood volume estimates from nonlinear and non-parametric methods significantly predicted increased microvascular hyperplasia. Abnormal microvasculature existed beyond the CE-lesion and was significantly reflected by increased blood volume from nonlinear, non-parametric, and scaled MR-signal analysis. This study provides histopathological support for both non-parametric and nonlinear post-processing of low flip-angle DSC-MRI for characterizing microvascular hyperplasia within GBM. Non-parametric analysis with a single gadolinium injection may be a particularly useful strategy clinically, as it requires less computational expense and limits gadolinium exposure.

Hypoxic cell waves around necrotic cores in glioblastoma: a biomathematical model and its therapeutic implications.

Glioblastoma is a rapidly evolving high-grade astrocytoma that is distinguished pathologically from lower grade gliomas by the presence of necrosis and microvascular hyperplasia. Necrotic areas are typically surrounded by hypercellular regions known as “pseudopalisades” originated by local tumor vessel occlusions that induce collective cellular migration events. This leads to the formation of waves of tumor cells actively migrating away from central hypoxia. We present a mathematical model that incorporates the interplay among two tumor cell phenotypes, a necrotic core and the oxygen distribution. Our simulations reveal the formation of a traveling wave of tumor cells that reproduces the observed histologic patterns of pseudopalisades. Additional simulations of the model equations show that preventing the collapse of tumor microvessels leads to slower glioma invasion, a fact that might be exploited for therapeutic purposes.