Purpose: We measured anterior cerebral artery (ACA)-middle cerebral artery (MCA) and posterior cerebral artery (PCA)-MCA pial filling on single-phase computed tomography angiograms (sCTAs) in acute ischemic stroke and correlate with the CTA-based Massachusetts General Hospital (MGH) and digital subtraction angiography (DSA)-based American Society of Interventional and Therapeutic Neuroradiology (ASITN) score. Methods: Patients with acute stroke and M1 MCA±intracranial internal carotid artery occlusion on baseline CTA were included. Baseline sCTA was assessed for phase of image acquisition. An evaluator assessed collaterals using the Calgary Collateral (CC) Score (measures pial arterial filling in ACA-MCA and PCA-MCA regions separately), the CTA-based MGH score, and on DSA using the ASITN score. Infarct volumes were measured on 24- to 48-hour magnetic resonance imaging/ computed tomography. Results: Of 106 patients, baseline sCTA was acquired in early arterial phase in 9.9%, peak arterial in 50.7%, equilibrium in 32.4%, early venous in 5.6%, and late venous in 1.4%. Variance in ACA-MCA collaterals explained only 32% of variance in PCA-MCA collaterals on the CC score (Spearman’s correlation coefficient rho [rho]=0.56). Correlation between ACA-MCA collaterals and the MGH score was strong (rho=0.8); correlation between PCA-MCA collaterals and this score was modest (rho=0.54). Correlation between ACA-MCA collaterals and the ASITN score was modest (n=53, rho=0.43); and correlation between PCA-MCA collaterals and ASITN score was poor (rho=0.33). Of the CTA-based scores, the CC Score (Akaike [AIC] 1022) was better at predicting follow-up infarct volumes than was the MGH score (AIC 1029). Conclusion: Collateral assessments in acute ischemic stroke are best done using CTA with temporal resolution and by assessing regional variability. ACA-MCA and MCA-PCA collaterals should be evaluated separately.

Glioblastoma (GBM) is an aggressive brain tumor that is resistant to conventional radiation and cytotoxic chemotherapies. We hypothesize that brain tumor initiating cells (BTICs), a subpopulation of treatment-resistant cells with stem cell properties cause tumor relapse and a subset of neural stem cell genes regulate BTIC self-renewal, driving GBM recurrence. We adapted the existing treatment protocol for adults with primary GBM for in vivo treatment of immunocompromised mice engrafted with GBMs. Post-chemoradiotherapy, the recovered GFP+GBMs were profiled for self-renewal and expression of critical stem cell genes. Using invitro and invivo gain-of-function/loss-of-function experiments, we investigated the regulatory functions of Bmi1 in primary neural stem & progenitor cells (NSPCs) and GBM tumor formation. Finally, global RNA-Seq profiling was performed to understand the consequences of Bmi1 dysregulation on target gene expression. GBM cells showed an increase in Bmi1 levels post-chemoradiotherapy, suggesting the presence of a treatment-refractory BTICs. GFP+cells extracted from treated xenografts had higher self-renewal and BTIC marker expression. Although treated mice responded to therapy, we observed tumor relapse with increased Bmi1 expression. Knockdown of Bmi1 diminished self-renewal and proliferation of GBM cells and delayed tumorigenesis, highlighting a critical role for Bmi1 in tumor maintenance. Conversely, over-expressing Bmi1 in NSPCs failed to initiate tumor formation in vivo. Using high-throughput sequencing data, we generated a map of signaling pathways dysregulated in GBM that may lead to tumor recurrence. Our data confirms the existence of a rare treatment-refractory BTIC population with enhanced self-renewal capacity that escapes therapy and drives tumor relapse.

Medulloblastoma (MB) is the most common malignant pediatric brain tumour, and is categorized into four molecular subgroups, with Group 3 MB having the worst prognosis due to the highest rate of metastatic dissemination and relapse. In this work, we describe the epigenetic regulator Bmi1 as a novel therapeutic target for treatment of recurrent Group 3 MB. Through comparative profiling of primary and recurrent MB, we show that Bmi1 defines a treatment-refractory cell population that is uniquely targetable by a novel class of small molecule inhibitors. We have optimized an in vivo mouse-adapted therapy model that has the advantage of generating recurrent, human, treatment-refractory MBs. Our preliminary studies showed that although chemoradiotherapy administered to mice engrafted with human MB showed reduction in tumour size, Bmi1 expression was enriched in the post-treatment residual tumour. Furthermore, we found that knockdown of Bmi1 in human recurrent MB cells decreases proliferation and self-renewing capacities of MB cells in vitro as well as both tumour size and extent of spinal leptomeningeal metastases in vivo. Oral administration of a potent Bmi1 inhibitor, PTC 028, resulted in a marked reduction in tumour burden and an increased survival in treatment cohort. Bmi1 inhibitors showed high specificity for MB cells and spared normal human neural stem cells, when treated with doses relevant for MB cells. As Group 3 medulloblastoma is often metastatic and uniformly fatal at recurrence, with no current or planned trials of targeted therapy, an efficacious agent such as Bmi1 inhibitor could be rapidly transitioned to clinical trials.

Glioblastoma (GBM), an aggressive primary adult brain tumor, is feared for its near uniformly fatal prognosis. Despite the use of aggressive treatment including surgical resection, radiotherapy and chemotherapy, the outcome of patients with GBM has failed to improve significantly. Numerous studies have implicated CD133+GBM subpopulation as driver of chemo- and radio-resistance. CD133 expression correlates with disease progression, recurrence, and poor overall survival of GBM patients. Here, we describe the preclinical evaluation of a recombinant CD133xCD3 bispecific T-cell engager (BiTE) antibody that redirects human polyclonal T cells to CD133+GBM cells, inducing very potent anti-tumor response. CD133-specific BiTE was constructed; with one arm recognizing the tumor antigen (CD133) while the second is specific to CD3 antigen. Using CD133high and CD133low primary GBM lines, we validated the binding of BiTEs to CD133+GBMs and CD3+T cells. In order to test the ability of BiTEs to functionally elicit CD133-specific cytotoxic responses in vitro, we performed killing assays. We observed CD133-specific BiTE mediated T cell activation and redirection to kill CD133-expressing GBM cells in a co-culture of T cells and GBM cells. The killing was more efficient in CD133high GBMs compared to CD133low GBMs, validating its specificity to target CD133+BTICs. Treatment with BiTEs yielded significant reductions in brain tumor burden in vivo. These data offers compelling evidence that BiTE-mediated cytotoxicity against treatment-resistant CD133+GBMs could provide a very potent, specific and can be a novel therapeutic strategy for GBM patients.

Medulloblastoma (MB), the most common malignant pediatric brain tumor, is categorized into four molecular subgroups. Given the high rate of metastatic dissemination at diagnosis and recurrence in Group 3 MBs, these patients have the worst clinical outcome with a 5-year survivorship of approximately 50%. By adapting the existing COG (Children’s Oncology Group) Protocol for children with newly diagnosed high-risk MB, for treatment of immuno-deficient mice intracranially engrafted with human MB brain tumour initiating cells we aim to identify and characterize the treatment-refractory cell population in Group 3 MBs. Mice were sacrificed at multiple time points during the course of tumor development and therapy: (i) at engraftment; (ii) post-radiation; (iii) post-radiation and chemotherapy; and (iv) at MB recurrence. MB cell populations recovered separately from brains and spines were comprehensively profiled for gene expression analysis, stem cell and molecular features to generate a global, comparative profile of MB cells through therapy. We report a higher expression of CD133, Sox2 and Bmi1 in addition to increased self-renewal capacity following chemoradiotherapy treatment. The enrichment map constructed from global gene expression analysis showed an increase in pathways regulating self-renewal, DNA repair and chemoresistance post-therapy despite the apparent decrease in tumour size and vascularity. Additionally, from gene expression at MB recurrence, we identified a list of genes that negatively correlate with survival in patients diagnosed with Group 3 MB. A differential genomic profile of the “treatment-responsive” tumors against those that fail therapy may contribute to discovery of novel therapeutic approaches for the most aggressive subgroup of MB.

Brain Metastases (BM) represent a leading cause of cancer mortality. While metastatic lesions contain subclones derived from their primary lesion, their functional characterization has been limited by a paucity of preclinical models accurately recapitulating the stages of metastasis. This work describes the isolation of a unique subset of metastatic stem-like cells from primary human patient samples of BM, termed brain metastasis initiating cells (BMICs). Utilizing these BMICs we have established a novel patient-derived xenograft (PDX) model of BM that recapitulates the entire metastatic cascade, from primary tumor initiation to micro-metastasis and macro-metastasis formation in the brain. We then comprehensively interrogated human BM to identify genetic regulators of BMICs using in vitro and in vivo RNA interference screens, and validated hits using both our novel PDX model as well as primary clinical BM specimens. We identified SPOCK1 and TWIST2 as novel BMIC regulators, where in our model SPOCK1 regulated BMIC self-renewal and tumor initiation, and TWIST2 specifically regulated cell migration from lung to brain. A prospective cohort of primary lung cancer specimens was used to establish that SPOCK1 and TWIST2 were only expressed in patients who ultimately developed BM, thus establishing both clinical and functional utility for these gene products. This work offers the first comprehensive preclinical model of human brain metastasis for further characterization of therapeutic targets, identification of predictive biomarkers, and subsequent prophylactic treatment of patients most likely to develop BM. By blocking this process, metastatic lung cancer would effectively become a localized, more manageable disease.

A total of 1244 egg-pods of 12 grasshopper species were collected — 26.7% were from low altitude hills, 23% from Swat valley, 22.1% from Punjab plains, 19.4% from Peshawar region and 8.8% from Potohar plateau. Aiolopus thalassinus was the dominant species comprising 27.7% of the total pods. This was followed by Shirakiacris shirakii (24.5%), Oxya multidentata (13.6%), Trilophidia annulata (9.7%), Spathosternum prasiniferum (9.7%), Atraetomorpha acutipennis(7.4%), Stenohippus sp. (3.8%) and Phlaeoba punteli (1.8%). Others were rare being less than 1%.

Egg-pod density was effected by several closely related factors. Mean annual temperatures (15.6 − 23.4°C) and average precipitation (310 − 2100 mm) of the localities were not the ultimate controlling factors. Parasitism however, appears to play an important role. Highest parasitism of Scelio spp. was 10% on the Potohar plateau where egg-pod population was lowest. A. thalassinus was the most preferred host(41.7%) followed by S. prasiniferum (35.4%), S. shirakii (14.6%), O. multidentata (8.3%), T. annulata and Stenohippus sp. (2.1% each). Soil-type (clay-loam, loam, sandy-loam) and type of vegetation(cultivated area, grasslands) also have a considerable bearing on the microclimate in which the insects live.