Search

Significance Brain metastases are among the most severe complications of systemic breast cancer, and overexpression of the human epidermal growth factor receptor 2 (HER2) in breast cancer cells increases the incidence of brain metastases in patients. In this study, we engineered the human-derived, tumor cell tropic neural stem cells LM-NSC008 (LM008) to continuously secrete antibodies against HER2. These anti-HER2 antibodies impaired tumor cell proliferation by inhibiting the PI3K-Akt signaling pathway in HER2+ breast cancer cells in vitro. Importantly, our results demonstrate that the therapeutic combinatorial regimen consisting of LM-NSC008 anti-HER2 antibody-secreting cells and the HER2 kinase inhibitor tucatinib provide therapeutic benefit and prolong survival in preclinical models of HER2+ breast cancer brain metastases., Brain metastases are a leading cause of death in patients with breast cancer. The lack of clinical trials and the presence of the blood–brain barrier limit therapeutic options. Furthermore, overexpression of the human epidermal growth factor receptor 2 (HER2) increases the incidence of breast cancer brain metastases (BCBM). HER2-targeting agents, such as the monoclonal antibodies trastuzumab and pertuzumab, improved outcomes in patients with breast cancer and extracranial metastases. However, continued BCBM progression in breast cancer patients highlighted the need for novel and effective targeted therapies against intracranial metastases. In this study, we engineered the highly migratory and brain tumor tropic human neural stem cells (NSCs) LM008 to continuously secrete high amounts of functional, stable, full-length antibodies against HER2 (anti-HER2Ab) without compromising the stemness of LM008 cells. The secreted anti-HER2Ab impaired tumor cell proliferation in vitro in HER2+ BCBM cells by inhibiting the PI3K-Akt signaling pathway and resulted in a significant benefit when injected in intracranial xenograft models. In addition, dual HER2 blockade using anti-HER2Ab LM008 NSCs and the tyrosine kinase inhibitor tucatinib significantly improved the survival of mice in a clinically relevant model of multiple HER2+ BCBM. These findings provide compelling evidence for the use of HER2Ab-secreting LM008 NSCs in combination with tucatinib as a promising therapeutic regimen for patients with HER2+ BCBM.

Simple Summary Disorders of bladder function can result from congenital spinal cord developmental defects and can remain in a significant number of patients despite surgical improvements to repair the primary defect. We studied the ability of bladder wall muscle from such patients to contract, a function essential to void collected urine and avoid urinary tract infections and potential damage to the kidneys. Tissue was taken when patients were several years old, at the time of surgical operations to improve bladder function. This tissue would otherwise have been discarded and was collected with the full ethical approval and consent of parents or guardians. We found that the ability of the bladder wall samples to contract was impaired and was generally stiffer; both of which would make it more difficult for the bladder to void urine. These functional changes were associated with a replacement of muscle with connective tissue (fibrosis). The experiments provide a pathway to devise strategies that might improve bladder function in these patients through reversal of the intrinsic tissue pathways that increase fibrosis. Abstract Neurogenic lower urinary tract (NLUT) dysfunction in paediatric patients can arise after congenital or acquired conditions that affect bladder innervation. With some patients, urinary tract dysfunction remains and is more difficult to treat without understanding the pathophysiology. We measured in vitro detrusor smooth muscle function of samples from such bladders and any association with altered Wnt-signalling pathways that contribute to both foetal development and connective tissue deposition. A comparator group was tissue from children with normally functioning bladders. Nerve-mediated and agonist-induced contractile responses and passive stiffness were measured. Histology measured smooth muscle and connective tissue proportions, and multiplex immunohistochemistry recorded expression of protein targets associated with Wnt-signalling pathways. Detrusor from the NLUT group had reduced contractility and greater stiffness, associated with increased connective tissue content. Immunohistochemistry showed no major changes to Wnt-signalling components except down-regulation of c-Myc, a multifunctional regulator of gene transcription. NLUT is a diverse term for several diagnoses that disrupt bladder innervation. While we cannot speculate about the reasons for these pathophysiological changes, their recognition should guide research to understand their ultimate causes and develop strategies to attenuate and even reverse them. The role of changes to the Wnt-signalling pathways was minor.

Despite an aggressive standard of care involving radiation therapy, temozolomide-based chemotherapy, and surgical resection, glioblastoma multiforme (GBM) continues to exhibit very high recurrence and mortality rates partly due to the highly plastic and heterogenous nature of the tumor. In recent years, activation of the immune system has emerged as a promising strategy in cancer therapies. However, despite recent successes in other fields, immunotherapeutic approaches continue to encounter challenges in GBM. In this review, we first discuss immunotherapies targeting the most well-studied immune checkpoint proteins, CTLA-4 and PD-1, followed by discussions on therapies targeting immune-stimulatory molecules and secreted metabolic enzymes. Finally, we address the major challenges with immunotherapy in GBM and the potential for combination and neoadjuvant immunotherapies to tip the scales in the fight against glioblastoma.

SUMMARYGlioblastoma (GBM) remains one of the least treatable types of cancer. Recent work highlights two key factors contributing to this resistant phenotype—cellular plasticity, the ability of GBM cells to adopt many phenotypes, and the microenvironment. Here, we provide evidence that Interleukin-8 (IL-8) plays a vital role in promoting cellular plasticity and cancer initiating cells (CICs) niche during anti-glioma chemotherapy. IL-8 expression is significantly elevated during chemotherapy, and immunohistochemical analysis of matched primary and recurrent patient GBM tissues revealed about 60% of recurrent tissues IL-8 expression is upregulated. In silico analysis of the TCGA data indicated that IL-8 signaling could promote epigenetic plasticity by altering the polycomb repressor complex activity. We are proposing that such regulation my promote epigenetic plasticity, which allows the GBM cells to adapt therapy and may promote therapeutic resistance. Our data show that IL-8 is a crucial microenvironmental factor involved in developing therapeutic adaptation and can be targeted in combination with conventional chemo-and radiotherapy to prevent disease recurrence.ABSTRACTEmerging evidence reveals enrichment of glioma initiating cells (GICs) following therapeutic intervention. This enrichment occurs partly by dedifferentiation of non-GICs to GICs within the tumor, which may contribute to therapeutic resistance and the generation of lethal recurrent tumors. To elucidate the molecular mechanisms governing therapy-induced cellular plasticity, we performed genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) and gene expression analysis (gene microarray analysis) during treatment with standard of care temozolomide (TMZ) chemotherapy. Analysis revealed significant enhancement of open chromatin marks in known astrocytic enhancers for Interleukin-8 (IL-8) loci as well as elevated expression during anti-glioma chemotherapy. The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project data demonstrated that IL-8 transcript expression is negatively correlated with GBM patient survival (p=0.001) and positively correlated with that of genes associated with the CIC phenotype such as KLF4, c-Myc and HIF2α (pin vivo (pin silico analysis of TCGA data, we examined the effect of therapy-induced IL-8 expression on the epigenomic landscape of GBM cells and observed increased trimethylation of H3K9 and H3K27. Our results show that IL-8 alters cellular plasticity and mediates alterations in histone status. These finding suggest that IL-8 signaling participates in regulating GBM adaptation to therapeutic stress and therefore represents a promising target for combination with conventional chemotherapy in order to limit GBM recurrence.

Class II antiarrhythmics or β-blockers are antisympathetic nervous system agents that act by blocking β-adrenoceptors. Despite their common clinical use, little is known about the effects of β-blockers on free intracellular calcium (Ca2+i), an important cytosolic second messenger and a key regulator of cell function. We investigated the role of four chemical analogs, commonly prescribed β-blockers (atenolol, metoprolol, propranolol, and sotalol), on Ca2+i release and whole-cell currents in mammalian cancer cells (PC3 prostate cancer and MCF7 breast cancer cell lines). We discovered that only propranolol activated free Ca2+i release with distinct kinetics, whereas atenolol, metoprolol, and sotalol did not. The propranolol-induced Ca2+i release was significantly inhibited by the chelation of extracellular calcium with ethylene glycol tetraacetic acid (EGTA) and by dantrolene, an inhibitor of the endoplasmic reticulum (ER) ryanodine receptor channels, and it was completely abolished by 2-aminoethoxydiphenyl borate, an inhibitor of the ER inositol-1,4,5-trisphosphate (IP3) receptor channels. Exhaustion of ER stores with 4-chloro-m-cresol, a ryanodine receptor activator, or thapsigargin, a sarco/ER Ca2+ ATPase inhibitor, precluded the propranolol-induced Ca2+i release. Finally, preincubation of cells with sotalol or timolol, nonselective blockers of β-adrenoceptors, also reduced the Ca2+i release activated by propranolol. Our results show that different β-blockers have differential effects on whole-cell currents and free Ca2+i release and that propranolol activates store-operated Ca2+i release via a mechanism that involves calcium-induced calcium release and putative downstream transducers such as IP3. The differential action of class II antiarrhythmics on Ca2+i release may have implications on the pharmacology of these drugs.

SUMMARY The mechanisms by which regulatory T cells (Tregs) migrate to and function within the hypoxic tumor microenvironment are unclear. Our studies indicate that specific ablation of hypoxia-inducible factor 1α (HIF-1α) in Tregs results in enhanced CD8+ T cell suppression versus wild-type Tregs under hypoxia, due to increased pyruvate import into the mitochondria. Importantly, HIF-1α-deficient Tregs are minimally affected by the inhibition of lipid oxidation, a fuel that is critical for Treg metabolism in tumors. Under hypoxia, HIF-1α directs glucose away from mitochondria, leaving Tregs dependent on fatty acids for mitochondrial metabolism within the hypoxic tumor. Indeed, inhibition of lipid oxidation enhances the survival of mice with glioma. Interestingly, HIF-1α-deficient-Treg mice exhibit significantly enhanced animal survival in a murine model of glioma, due to their stymied migratory capacity, explaining their reduced abundance in tumor-bearing mice. Thus HIF-1α acts as a metabolic switch for Tregs between glycolytic-driven migration and oxidative phosphorylation-driven immunosuppression., Graphical Abstract, In Brief Miska et al. demonstrate that regulatory T cell (Treg)-specific depletion of HIF-1α promotes enhanced immune suppression at the cost of migration under hypoxic conditions. Within the hypoxic brain-tumor environment, Tregs are uniquely able to metabolize extracellular free fatty acids to promote their immunosuppressive functionality, which can be targeted in vivo.

Glioblastoma is an incredibly aggressive primary brain tumor that is universally lethal due to 100% recurrence. Recent research has pointed to the existence of a population of cells that possess stem cell-like characteristics that are resistant to conventional therapy and can initiate recurrence. Our laboratory, along with others, has demonstrated that this stem-like state is plastic and can be acquired by otherwise differentiated GBM cells exposed to different stress, including stress generated by chemotherapy. Our Initial investigation indicated that Polycomb group protein EZH2 is critical for therapeutic stress-induced cellular plasticity. Further investigation revealed that the mechanisms of EZH2-mediated cellular plasticity are partly governed by a novel downstream target ARL13B, a member of the ADP-ribosylation factor-like family protein critical for cilia formation and maintenance. ARl13B removal significantly reduced different stemness factors such as nestin, SOX2, and most importantly, sensitized different subtypes of patient-derived xenograft lines to temozolomide-based chemotherapy both in vitro and in vivo (p Citation Format: Atique U. Ahmed, Jack M. Shireman, Fatemeh Atash, Gina Lee, Eunus S. Ali, Miranda R. Saathoff, Cheol H. Park, Sol Savchuk, Shivani Baisiwala, Jason Miska, Maciej S. Lesniak, C. David James, Roger Stupp, Priya Kumthekar, Craig M. Horbinski, Issam Ben-Sahra. Targeting cellular plasticity-driven metabolic adaptation to overcome chemoresistance in GBM [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr PR011.

This abstract is being presented as a short talk in the scientific program. A full abstract is available in the Proffered Abstracts section (PR011) of the Conference Proceedings. Citation Format: Atique U. Ahmed, Jack M. Shireman, Fatemeh Atash, Gina Lee, Eunus S. Ali, Miranda R. Saathoff, Cheol H. Park, Sol Savchuk, Shivani Baisiwala, Jason Miska, Maciej S. Lesniak, C. David James, Roger Stupp, Priya Kumthekar, Craig M. Horbinski, Issam Ben-Sahra. Targeting cellular plasticity-driven metabolic adaptation to overcome chemoresistance in GBM [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B034.

Background and AimsLiver cancer comprises of benign or malignant tumors including hepatocellular carcinoma (HCC), cholangiocarcinoma (CC), hepatoblastoma (HB), and other rarer tumor types. There is evidence of aberrant Wnt signaling during initiation and progression of HCC, CC and HB.MethodsWe investigated the expression of Wnt/β-catenin transcription related proteins, Cyclin D1, c-Myc, Fra-1 and Pygo-1, in human liver tumors by using an unbiased, quantitative immunohistochemical (qIHC) approach.ResultsSemi-automated, unbiased quantitation of individual proteins revealed reduced expression of Cyclin D1 and Pygo-1 in CC (P < 0.0001 and P < 0.01, respectively) and HB (P < 0.05 and P < 0.01, respectively) compared to normal liver (NL). Receiver operating characteristic curves showed Cyclin D1 as a putative marker for CC (AUC > 0.8) that discriminates CC from both NL and HCC (P ≤ 0.0001), and Pygo-1 (AUC > 0.7) as a marker for both CC and HCC (P < 0.01) compared to NL. Combining Cyclin D1/Pygo-1 and applying a logistic regression model further improved the diagnostic potential (classifying 84% of NL and CC cases, P < 0.0001). Quantitative co-localisation of tissue samples simultaneously labeled with the four biomarkers, indicated that co-localisation of both Pygo-1/Fra-1 and c-Myc/Fra-1 was also significantly changed in CC and HCC (P < 0.0001) vs NL. Additionally, co-localisation of Pygo-1/Fra-1, in particular, could also distinguish CC from HCC (P < 0.05).ConclusionOur results indicate that measurement of Wnt signaling markers could be used to stratify liver cancer.

Equine penile squamous cell carcinoma (EpSCC) is a relatively common cutaneous neoplasm with a poor prognosis. In this study, we aimed to determine the protein expression and colocalisation of FRA1, c-Myc, Cyclin D1, and MMP7 in normal (NT), tumour (T), hyperplastic epidermis and/or squamous papilloma (Hyp/Pap), poorly-differentiated (PDSCC), or well-differentiated (WDSCC) EpSCC using a tissue array approach. Further objectives were to correlate protein expression to (i) levels of inflammation, using a convolutional neural network (ii) equine papillomavirus 2 (EcPV2) infection, detected using PCR amplification. We found an increase in expression of FRA1 in EpSCC compared to NT samples. c-Myc expression was higher in Hyp/Pap and WDSCC but not PDSCC whereas MMP7 was reduced in WDSCC compared with NT. There was a significant increase in the global intersection coefficient (GIC) of FRA1 with MMP7, c-Myc, and Cyclin D1 in EpSCC. Conversely, GIC for MMP7 with c-Myc was reduced in EpSCC tissue. Inflammation was positively associated with EcPV2 infection in both NT and EpSCC but not Hyp/Pap. Changes in protein expression could be correlated with EcPV2 for Cyclin D1 and c-Myc. Our results evaluate novel biomarkers of EpSCC and a putative correlation between the expression of biomarkers, EcPV2 infection and inflammation.

Summary Introduction Posterior urethral valves (PUV) is the most common cause of congenital bladder outflow obstruction with persistent lower urinary tract and renal morbidities. There is a spectrum of functional bladder disorders ranging from hypertonia to bladder underactivity, but the aetiology of these clinical conditions remains unclear. Aims and objectives We tested the hypothesis that replacement of detrusor muscle with non-muscle cells and excessive deposition of connective tissue is an important factor in bladder dysfunction with PUV. We used isolated detrusor samples from children with PUV and undergoing primary or secondary procedures in comparison to age-matched data from children with functionally normal bladders. In vitro contractile properties, as well as passive stiffness, were measured and matched to histological assessment of muscle and connective tissue. We examined if a major pathway for fibrosis was altered in PUV tissue samples. Methods Isometric contractions were measured in vitro in response to either stimulation of motor nerves to detrusor or exposure to cholinergic and purinergic receptor agonists. Passive mechanical stiffness was measured by rapid stretching of the tissue and recording changes to muscle tension. Histology measured the relative amounts of detrusor muscle and connective tissue. Multiplex quantitative immunofluorescence labelling using five epitope markers was designed to determine cellular pathways, in particular the Wnt-signalling pathway, responsible for any changes to excessive deposition of connective tissue. Results and Discussion PUV tissue showed equally reduced contractile function to efferent nerve stimulation or exposure to contractile agonists. Passive muscle stiffness was increased in PUV tissue samples. The smooth muscle:connective tissue ratio was also diminished and mirrored the reduction of contractile function and the increase of passive stiffness. Immunofluorescence labelling showed in PUV samples increased expression of the matrix metalloproteinase, MMP-7; as well as cyclin-D1 expression suggesting cellular remodelling. However, elements of a fibrosis pathway associated with Wnt-signalling were either reduced (β-catenin) or unchanged (c-Myc). The accumulation of extracellular matrix, containing collagen, will contribute to the reduced contractile performance of the bladder wall. It will also increase tissue stiffness that in vivo would lead to reduced filling compliance. Conclusions Replacement of smooth muscle with fibrosis is a major contributory factor in contractile dysfunction in the hypertonic PUV bladder. This suggests that a potential strategy to restore normal contractile and filling properties is development of the effective use of antifibrotic agents. Download : Download high-res image (132KB) Download : Download full-size image Summary Figure .

Key points: Wnt ligands belonging to both canonical and non-canonical Wnt pathways regulate membrane potential signifying a very early event in the signal transduction. Wnts activate K + currents by elevating intracellular Ca 2+ and trigger Ca 2+ release from intracellular stores. Control of potential by Wnt ligands has significant implications for gene transcription and opens up a novel avenue to interfere with this critical pathway. Abstract: The Wnt signalling network determines gene transcription with free intracellular Ca 2+ (Ca 2+ i) and β-catenin as major intracellular signal transducers. Despite its critical importance during development and disease, many of the basic mechanisms of Wnt signal activation remain unclear. Here we show by single cell recording and simultaneous Ca 2+ i imaging in mammalian prostate cancer cells that an early step in the signal cascade is direct action on the cell membrane potential. We show that Wnt ligands 5A, 9B and 10B rapidly hyperpolarized the cells by activating K + current by Ca 2+ release from intracellular stores. Medium-throughput multi-well recordings showed responses to Wnts at concentrations of 2 nm. We identify a putative target for early events as a TRPM channel. Wnts thus act as ligands for ion channel activation in mammalian cells and membrane potential is an early indicator of control of transcription.

Motivation Tissue array (TA) staining, combined with whole slide imaging (WSI) methods facilitate discovery of biomarkers for diagnosis, prognostication and disease stratification. A key impediment in TA WSI analysis is handling missing tissue and artefacts when identifying tissue cores before quantitative, standardized downstream analysis. There is a need for an open access, user friendly, integrated analysis of the WSI generated using TAs in clinical and scientific research laboratories. Results We have developed QuArray (Quantitative Array Application) for image export and signal analysis of TAs using WSI. The application input is a WSI and a corresponding TA configuration file. QuArray identifies and exports core images and analyses chromogen staining in a simple graphical user interface. Output data is saved to file for further analysis including indexed data. Availabilityand implementation Available for download from https://github.com/c-arthurs/QuArray under an MIT licence. Supplementary information Supplementary data are available at Bioinformatics online.

It remains incumbent on researchers to conceive novel treatments for the most common primary malignancy of the brain in adults, glioblastoma multiforme (GBM), as the standard of care for patients today fails to yield a median survival beyond two years following diagnosis. Recent studies have tended towards appreciating the cellular heterogeneity of GBM tumors, focusing on the subpopulation of highly plastic glioblastoma stem cells (GSCs). In the November 2016 issue of Cell, Hu and colleagues developed a de nova GBM model derived from immortalized neural stem cells and, using this model, they demonstrated that GSCs can generate CD133+/CD144+ cells with endothelial cell-like characteristics. Contrasts between the epigenetic state and gene expression level before and after oncogenic transformation of this utilized de novo model for GBM implicated WNT5A, which has been previously shown to play a role in endothelial cell proliferation and migration via non-canonical Wnt signaling, as a mediator of the process. The transdifferentiation was accompanied by alterations in the histone marks at the gene loci of WNT5A, and its transcription factors PAX6 and DXL5. The authors hypothesize that activation of AKT, an aberration of the RTK/PTEN/PI3K pathway observed in the majority of GBM cases, triggers these epigenetic changes causing WNT5A expression. This phenomenon is of obvious clinical significance, as it provides an insight into how GBM may circumvent therapies targeting angiogenesis to achieve the neovascularization required to sustain invasive growth. The unveiling of this atypical differentiation process also raises questions about its interaction with the radiotherapy and chemotherapy commonly used to counter GBM progression. Here, we review the recent efforts to understand the complex mechanisms behind the plasticity of GSCs.

Free intracellular calcium ([Ca2+]i), in addition to being an important second messenger, is a key regulator of many cellular processes including the cell membrane potential, proliferation and apoptosis. In many cases, the mobilization of [Ca2+]i is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on [Ca2+]i release, by ratiometric calcium imaging. We show that six such modulators (Amiodarone (Ami), Dofetilide (Dof), Furosemide (Fur), Minoxidil (Min), Loxapine (Lox), and Nicorandil (Nic)) initiate release of [Ca2+]i in prostate and breast cancer cell lines, PC3 and MCF7, respectively. Whole cell currents in PC3 cells were inhibited by the compounds tested in patch clamp experiments in a concentration dependent manner. In all cases [Ca2+]i was increased by modulator concentrations comparable to those used clinically. The increase in [Ca2+]i in response to Ami, Fur, Lox and Min was reduced, significantly (p

Increasing evidence exposes a subpopulation of cancer cells, known as cancer stem cells (CSCs), to be critical for the progression of several human malignancies, including glioblastoma multiforme. CSCs are highly tumorigenic, capable of self-renewal, and resistant to conventional therapies, and thus considered to be one of the key contributors to disease recurrence. To elucidate the poorly understood evolutionary path of tumor recurrence and the role of CSCs in this process, we developed patient-derived xenograft glioblastoma recurrent models induced by anti-glioma chemotherapy, temozolomide. In this model, we observed a significant phenotypic shift towards an undifferentiated population. We confirmed these findings in vitro as sorted CD133-negative populations cultured in differentiation-forcing media were found to acquire CD133 expression following chemotherapy treatment. To investigate this phenotypic switch at the single-cell level, glioma stem cell (GSC)-specific promoter-based reporter systems were engineered to track changes in the GSC population in real time. We observed the active phenotypic and functional switch of single non-stem glioma cells to a stem-like state and that temozolomide therapy significantly increased the rate of single-cell conversions. Importantly, we showed the therapy-induced hypoxia-inducible factors (HIF) 1α and HIF2α play key roles in allowing non-stem glioma cells to acquire stem-like traits, as the expression of both HIFs increase upon temozolomide therapy and knockdown of HIFs expression inhibits the interconversion between non-stem glioma cells and GSCs post-therapy. On the basis of our results, we propose that anti-glioma chemotherapy promotes the accumulation of HIFs in the glioblastoma multiforme cells that induces the formation of therapy-resistant GSCs responsible for recurrence. Mol Cancer Ther; 15(12); 3064–76. ©2016 AACR.

Spontaneous canine (Canis lupus) oligodendroglioma (ODG) holds tremendous potential as an immunocompetent large animal model of human malignant gliomas (MG). However, the feasibility of utilizing this model in pre-clinical studies depends on a thorough understanding of the similarities and differences of the molecular pathways associated with gliomas between the two species. We have previously shown that canine ODG has an immune landscape and expression pattern of commonly described oncogenes similar to that of human MG. In the current study, we performed a comprehensive analysis of canine ODG RNAseq data from 4 dogs with ODG and 2 normal controls to identify highly dysregulated genes in canine tumors. We then evaluated the expression of these genes in human MG using Xena Browser, a publicly available database. STRING-database inquiry was used in order to determine the suggested protein associations of these differentially expressed genes as well as the dysregulated pathways commonly enriched by the protein products of these genes in both canine ODG and human MG. Our results revealed that 3,712 (23%) of the 15,895 differentially expressed genes demonstrated significant up- or downregulation (log2-fold change > 2.0). Of the 3,712 altered genes, ~50% were upregulated (n = 1858) and ~50% were downregulated (n = 1854). Most of these genes were also found to have altered expression in human MG. Protein association and pathway analysis revealed common pathways enriched by members of the up- and downregulated gene categories in both species. In summary, we demonstrate that a similar pattern of gene dysregulation characterizes both human MG and canine ODG and provide additional support for the use of the canine model in order to therapeutically target these common genes. The results of such therapeutic targeting in the canine model can serve to more accurately predict the efficacy of anti-glioma therapies in human patients.

Optimal treatment of breast cancer brain metastases (BCBM) is often hampered by limitations in diagnostic abilities. Developing innovative tools for BCBM diagnosis is vital for early detection and effective treatment. In this study we explored the advances in trial for the diagnosis of BCBM, with review of the literature. On May 8, 2019, we searched ClinicalTrials.gov for interventional and diagnostic clinical trials involving BCBM, without limiting for date or location. Information on trial characteristics, experimental interventions, results, and publications were collected and analyzed. In addition, a systematic review of the literature was conducted to explore published studies related to BCBM diagnosis. Only 9 diagnostic trials explored BCBM. Of these, 1 trial was withdrawn because of low accrual numbers. Three trials were completed; however, none had published results. Modalities in trial for BCBM diagnosis entailed magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), PET-CT, nanobodies, and circulating tumor cells (CTCs), along with a collection of novel tracers and imaging biomarkers. MRI continues to be the diagnostic modality of choice, whereas CT is best suited for acute settings. Advances in PET and PET-CT allow the collection of metabolic and functional information related to BCBM. CTC characterization can help reflect on the molecular foundations of BCBM, whereas cell-free DNA offers new genetic material for further exploration in trials. The integration of machine learning in BCBM diagnosis seems inevitable as we continue to aim for rapid and accurate detection and better patient outcomes.

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor affecting adults, with a median survival of approximately 21 months. One key factor underlying the limited efficacy of current treatment modalities is the remarkable plasticity exhibited by GBM cells, which allows them to effectively adapt to changes induced by anticancer therapeutics. Moreover, GBM tumors are highly vascularized with aberrant vessels that complicate the delivery of antitumor agents. Recent research has demonstrated that GBM cells have the ability to transdifferentiate into endothelial cells (ECs), illustrating that GBM cells may use plasticity in concert with vascularization leading to the creation of tumor-derived blood vessels. The mechanism behind this transdifferentiation, however, remains unclear. Here, we show that treatment with temozolomide (TMZ) chemotherapy induces time-dependent expression of markers for glioma stem cells (GSCs) and immature and mature ECs. In addition, GBM tumors growing as orthotopic xenografts in nude mice showed increased expression of GSC and EC markers after TMZ treatment. Ex vivo FACS analysis showed the presence of immature and mature EC populations. Furthermore, immunofluorescence analysis revealed increased tumor-derived vessels in TMZ-recurrent tumors. Overall, this study identifies chemotherapeutic stress as a new driver of transdifferentiation of tumor cells to endothelial cells and highlights cellular plasticity as a key player in therapeutic resistance and tumor recurrence.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer patients is associated with increased incidence of breast cancer brain metastases (BCBM), but the mechanisms underlying this phenomenon remain unclear. Here, to identify brain-predominant genes critical for the establishment of BCBM, we conducted an in silico screening analysis and identified that increased levels of fatty acid-binding protein 7 (FABP7) correlate with a lower survival and higher incidence of brain metastases in breast cancer patients. We validated these findings using HER2+ BCBM cells compared with parental breast cancer cells. Importantly, through knockdown and overexpression assays, we characterized the role of FABP7 in the BCBM process in vitro and in vivo. Our results uncover a key role of FABP7 in metabolic reprogramming of HER2 + breast cancer cells, supporting a glycolytic phenotype and storage of lipid droplets that enable their adaptation and survival in the brain microenvironment. In addition, FABP7 is shown to be required for upregulation of key metastatic genes and pathways, such as integrins-Src and VEGFA, and for the growth of HER2+ breast cancer cells in the brain microenvironment in vivo. Together, our results support FABP7 as a potential target for the treatment of HER2+ BCBM.

In many aggressive cancers, such as glioblastoma multiforme, progression is enabled by local immunosuppression driven by the accumulation of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). However, the mechanistic details of how Tregs and MDSCs are recruited in various tumors are not yet well understood. Here we report that macrophages and microglia within the glioma microenvironment produce CCL2, a chemokine that is critical for recruiting both CCR4+ Treg and CCR2+Ly-6C+ monocytic MDSCs in this disease setting. In murine gliomas, we established novel roles for tumor-derived CCL20 and osteoprotegerin in inducing CCL2 production from macrophages and microglia. Tumors grown in CCL2-deficient mice failed to maximally accrue Tregs and monocytic MDSCs. In mixed-bone marrow chimera assays, we found that CCR4-deficient Treg and CCR2-deficient monocytic MDSCs were defective in glioma accumulation. Furthermore, administration of a small-molecule antagonist of CCR4 improved median survival in the model. In clinical specimens of glioblastoma multiforme, elevated levels of CCL2 expression correlated with reduced overall survival of patients. Finally, we found that CD163-positive infiltrating macrophages were a major source of CCL2 in glioblastoma multiforme patients. Collectively, our findings show how glioma cells influence the tumor microenvironment to recruit potent effectors of immunosuppression that drive progression. Cancer Res; 76(19); 5671–82. ©2016 AACR.

Summary The challenges to effective drug delivery to brain tumors are twofold: (1) there is a lack of non-invasive methods of local delivery and (2) the blood-brain barrier limits systemic delivery. Intranasal delivery of therapeutics to the brain overcomes both challenges. In mouse model of malignant glioma, we observed that a small fraction of intranasally delivered neural stem cells (NSCs) can migrate to the brain tumor site. Here, we demonstrate that hypoxic preconditioning or overexpression of CXCR4 significantly enhances the tumor-targeting ability of NSCs, but without altering their phenotype only in genetically modified NSCs. Modified NSCs deliver oncolytic virus to glioma more efficiently and extend survival of experimental animals in the context of radiotherapy. Our findings indicate that intranasal delivery of stem cell-based therapeutics could be optimized for future clinical applications, and allow for safe and repeated administration of biological therapies to brain tumors and other CNS disorders., Graphical Abstract Image 1, Highlights • Intranasal delivery of NSCs is a promising platform for glioma therapy • Hypoxia or CXCR4 overexpression enhances NSC migration to glioma • Oncolytic viruses loaded in CXCR4-enhanced NSCs improve animal survival • Non-invasive intranasal NSC-based therapies warrant clinical translation, In this report, Balyasnikova and colleagues demonstrate that hypoxic preconditioning or overexpression of CXCR4 in neural stem cells enhances intranasal delivery of oncolytic virus to irradiated glioma, resulting in survival benefits for experimental animals. These findings indicate that intranasal stem cell-based oncolytic virotherapy could be optimized for future clinical applications in the context of brain tumors.

Glioblastoma (GBM) is one of the most aggressive and lethal tumor types. Evidence continues to accrue indicating that the complex relationship between GBM and the brain microenvironment contributes to this malignant phenotype. However, the interaction between GBM and neurotransmitters, signaling molecules involved in neuronal communication, remains incompletely understood. Here we examined, in both sexes of humans and mice, how the monoamine dopamine influences GBM cells. We demonstrate that GBM cells express DRD2, with elevated expression in the glioma-initiating cell (GIC) population. Stimulation of DRD2 caused neuron-like depolarization exclusively in GICs. In addition, long-term activation of DRD2 heightened the sphere-forming capacity of GBM cells as well as tumor engraftment efficiency. Mechanistic investigation revealed that DRD2 signaling activates the hypoxia response and functionally alters metabolism. Finally, we found that GBM cells synthesize and secrete dopamine themselves, suggesting a potential autocrine mechanism. These results identify dopamine signaling as a potential therapeutic target in GBM and further highlight neurotransmitters as a key feature of the pro-tumor microenvironment.Significance StatementThis work offers critical insight into the role of the neurotransmitter dopamine in the progression of GBM. We show that dopamine induces specific changes in the state of tumor cells, augmenting their growth and shifting them to a more stem-cell like state. Further, we show that dopamine can alter the metabolic behavior of GBM cells, increasing glycolysis. Finally, we show that GBM cells, including tumor samples from patients, can synthesize and secrete dopamine, suggesting an autocrine signaling process underlying these results. These results describe a novel connection between neurotransmitters and brain cancer, further highlighting the critical influence of the brain milieu on GBM.

Background Breast cancer brain metastases (BCBM) are the final frontier in neuro-oncology for which more efficacious therapies are required. In this work, we explore clinical trials in BCBM, and determine the shortcomings in the development of new BCBM therapies to shed light on potential areas for enhancement. Methods On July 9, 2018, we searched ClinicalTrials.gov for all interventional and therapeutic clinical trials involving BCBM, without limiting for date or location. Information on trial characteristics, including phase, status, start and end dates, study design, primary endpoints, selection criteria, sample size, experimental interventions, results, and publications were collected and analyzed. Results Fifty-three trials fulfilled the selection criteria. Median trial duration across phases ranged between 3 and 6 years. More than half of the trials were conducted in the United States. Although 94% of the trials were in early phases (I-II), 20% of patients were in phase III trials. Two phase III trials were anteceded by phase II trials that were non-randomized; one reported positive results. Approximately one-third of the trials were completed, whereas 23% of trials were terminated early; mostly due to inadequate enrollment. Only 13% of all trials and 22% of completed trials had published results directly linked to their primary outcomes. Conclusions The low number of trials and accrual numbers, the lack of diversity, and the scarcity of published results represent the main troubles in clinical BCBM research. Optimization of BCBM trials is necessary to achieve effective therapies.

Summary Introduction Bladder exstrophy is a congenital anomaly involving foetal exposure and protrusion of the open bladder through an incomplete lower abdominal wall. Techniques to surgically correct exstrophy after birth have greatly improved, but it still presents a major challenge to achieve continence and a good quality of life for patients and their families as the pathophysiology of bladder dysfunction is unknown. Objectives A multimodal approach was used to characterise the histological and biomechanical properties of exstrophy detrusor. These were correlated with myocyte responses to agonists and an evaluation of developmental signalling pathways to evaluate the cause of bladder dysfunction in exstrophy. Study design Detrusor muscle specimens were obtained during corrective surgery from four exstrophy groups: neonatal (1–3 days, n = 8), younger children (7 months–5 years, n = 13) and older children (8–14 years, n = 11) undergoing secondary procedures and cloacal exstrophy (16 days–9 years, n = 9); control specimens were obtained from children (3 months–9 years, n = 14) undergoing surgery for other pathologies but with normal bladder function. Five lines of experiments were undertaken: measurement of connective tissue to detrusor muscle ratio, contractile responses to electrical and agonist stimulation; in vitro biomechanical stiffness, intracellular Ca2+ responses to contractile agonists and immunohistochemistry for proteins (MMP-7, cyclinD1, β-catenin and c-myc) involved in fibrosis generation. Exstrophy data were compared with those from the control group. Results Exstrophy tissue demonstrated reduced smooth muscle compared with connective tissue, reduced contractile responses and greater mechanical stiffness. However, intracellular Ca2+ responses to agonists were maintained. These changes were greatest in neonatal and cloacal exstrophy samples and least in those from older paediatric bladders. Immunolabelled MMP-7, β-catenin and c-myc were reduced in exstrophy samples. Discussion These results highlight the reality that newborns with exstrophy have significantly reduced compliance and bladder underactivity, which may persist or return to normal values with surgery and age. The primary cause of underactivity is increased connective tissue in relation to detrusor muscle; however, detrusor myocyte function remains normal. Finally, the increase of the smooth muscle content in the paediatric bladder group indicates a remodelling response of the bladder to surgical correction and time. Excess gestational fibrosis is associated with changed expression of key proteins in the Wnt-signalling pathway, a potential aetiological factor and therapeutic target. Conclusion Results point to connective tissue deposition as the primary pathological process that determines bladder function with normal myocyte function. Future research that reduces connective tissue deposition may lead to improvement in outcomes for these children. Download : Download high-res image (124KB) Download : Download full-size image

Glioblastoma (GBM) is one of the most aggressive and lethal tumor types. Evidence continues to accrue indicating that the complex relationship between GBM and the brain microenvironment contributes to this malignant phenotype. However, the interaction between GBM and neurotransmitters, signaling molecules involved in neuronal communication, remains incompletely understood. Here we examined, using human patient-derived xenograft lines, how the monoamine dopamine influences GBM cells. We demonstrate that GBM cells express dopamine receptor 2 (DRD2), with elevated expression in the glioma-initiating cell (GIC) population. Stimulation of DRD2 caused a neuron-like hyperpolarization exclusively in GICs. In addition, long-term activation of DRD2 heightened the sphere-forming capacity of GBM cells, as well as tumor engraftment efficiency in both male and female mice. Mechanistic investigation revealed that DRD2 signaling activates the hypoxia response and functionally alters metabolism. Finally, we found that GBM cells synthesize and secrete dopamine themselves, suggesting a potential autocrine mechanism. These results identify dopamine signaling as a potential therapeutic target in GBM and further highlight neurotransmitters as a key feature of the pro-tumor microenvironment.SIGNIFICANCE STATEMENTThis work offers critical insight into the role of the neurotransmitter dopamine in the progression of GBM. We show that dopamine induces specific changes in the state of tumor cells, augmenting their growth and shifting them to a more stem-cell like state. Further, our data illustrate that dopamine can alter the metabolic behavior of GBM cells, increasing glycolysis. Finally, this work demonstrates that GBM cells, including tumor samples from patients, can synthesize and secrete dopamine, suggesting an autocrine signaling process underlying these results. These results describe a novel connection between neurotransmitters and brain cancer, further highlighting the critical influence of the brain milieu on GBM.

Radiation became a pillar of oncologic treatment in the last century and provided a powerful and effective locoregional treatment of solid malignancies. After achieving some of the first cures in lymphomas and skin cancers, it assumed a key role in the curative treatment of epithelioid malignancies. Despite success across a variety of histologic types, glioblastoma (GBM), the most common primary brain tumor afflicting adults, remains ultimately resistant to current radiation strategies. While GBMs demonstrate an initial response, recurrence is essentially universal and fatal and typically reoccur in the areas that received the most intense radiation. Glioma stem cells (GSCs), a subpopulation of tumor cells with expression profiles similar to neural stem cells and marked self-renewal capacities, have been shown to drive tumor recurrence and preclude curative radiotherapy. Recent research has shown that these cells have enhanced DNA repair capacity and elevated resistance to cytotoxic ion fluxes and escape multimodality therapies. We will analyze the current understanding of GSCs and radiation by highlighting key discoveries probing their ability to withstand radiotherapy. We then speculate on novel mechanisms by which GSC can be made sensitive to or specifically targeted by radiation therapy.

Oncolytic virotherapy for cancer is an innovative therapeutic option where the ability of a virus to promote cell lysis is harnessed and reprogrammed to selectively destroy cancer cells. Such treatment modalities exhibited antitumor activity in preclinical and clinical settings and appear to be well tolerated when tested in clinical trials. However, the clinical success of oncolytic virotherapy has been significantly hampered due to the inability to target systematic metastasis. This is partly due to the inability of the therapeutic virus to survive in the patient circulation, in order to target tumors at distant sites. An early study from various laboratories demonstrated that cells infected with oncolytic virus can protect the therapeutic payload form the host immune system as well as function as factories for virus production and enhance the therapeutic efficacy of oncolytic virus. While a variety of cell lineages possessed potential as cell carriers, copious investigation has established stem cells as a very attractive cell carrier system in oncolytic virotherapy. The ideal cell carrier desire to be susceptible to viral infection as well as support viral infection, maintain immunosuppressive properties to shield the loaded viruses from the host immune system, and most importantly possess an intrinsic tumor homing ability to deliver loaded viruses directly to the site of the metastasis—all qualities stem cells exhibit. In this review, we summarize the recent work in the development of stem cell-based carrier for oncolytic virotherapy, discuss the advantages and disadvantages of a variety of cell carriers, especially focusing on why stem cells have emerged as the leading candidate, and finally propose a future direction for stem cell-based targeted oncolytic virotherapy that involves its establishment as a viable treatment option for cancer patients in the clinical setting.

The dismal clinical context of advanced-grade glioma demands the development of novel therapeutic strategies with direct patient impact. Adenovirus-mediated virotherapy represents a potentially effective approach for glioma therapy. In this research, we generated a novel glioma-specific adenovirus by instituting more advanced genetic modifications that can maximize the efficiency and safety of therapeutic adenoviral vectors. In this regard, a glioma-specific targeted fiber was developed through the incorporation of previously published glioma-specific, phage-panned peptide (VWT peptide) on a fiber fibritin-based chimeric fiber, designated as "GliomaFF." We showed that the entry of this virus was highly restricted to glioma cells, supporting the specificity imparted by the phage-panned peptide. In addition, the stability of the targeting moiety presented by fiber fibritin structure permitted greatly enhanced infectivity. Furthermore, the replication of this virus was restricted in glioma cells by controlling expression of the E1 gene under the activity of the tumor-specific survivin promoter. Using this approach, we were able to explore the combinatorial efficacy of various adenoviral modifications that could amplify the specificity, infectivity, and exclusive replication of this therapeutic adenovirus in glioma. Finally, virotherapy with this modified virus resulted in up to 70% extended survival in an in vivo murine glioma model. These data demonstrate that this novel adenoviral vector is a safe and efficient treatment for this difficult malignancy.

The treatment of human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer has been revolutionized by trastuzumab. However, longer survival of these patients now predisposes them to forming HER2 positive brain metastases, as the therapeutic antibodies cannot cross the blood brain barrier. The current oncologic repertoire does not offer a rational, nontoxic targeted therapy for brain metastases. In this study, we used an established human neural stem cell line, HB1.F3 NSCs and generated a stable pool of cells secreting a high amount of functional full-length anti-HER2 antibody, equivalent to trastuzumab. Anti-HER2Ab secreted by the NSCs (HER2Ab-NSCs) specifically binds to HER2 overexpressing human breast cancer cells and inhibits PI3K-Akt signaling. This translates to HER2Ab-NSC inhibition of breast cancer cell growth in vitro. Preclinical in vivo experiments using HER2Ab overexpressing NSCs in a breast cancer brain metastases (BCBM) mouse model demonstrate that intracranial injection of HER2Ab-NSCs significantly improves survival. In effect, these NSCs provide tumor localized production of HER2Ab, minimizing any potential off-target side effects. Our results establish HER2Ab-NSCs as a novel, nontoxic, and rational therapeutic approach for the successful treatment of HER2 overexpressing BCBM, which now warrants further preclinical and clinical investigation. Stem Cells 2015;33:2985–2994

Glioma stem cells (GSCs) constitute a slow-dividing, small population within a heterogeneous glioblastoma. They are able to self-renew, recapitulate a whole tumor, and differentiate into other specific glioblastoma multiforme (GBM) subpopulations. Therefore, they have been held responsible for malignant relapse after primary standard therapy and the poor prognosis of recurrent GBM. The failure of current therapies to eliminate specific GSC subpopulations has been considered a major factor contributing to the inevitable recurrence in GBM patients after treatment. Here, we discuss the molecular mechanisms of chemoresistance of GSCs and the reasons why complete eradication of GSCs is so difficult to achieve. We will also describe the targeted therapies currently available for GSCs and possible mechanisms to overcome such chemoresistance and avoid therapeutic relapse.

Brain metastases occur in about 10% to 30% of breast cancer patients, which culminates in a poor prognosis. It is, therefore, critical to understand the molecular mechanisms underlying brain metastatic processes to identify relevant targets. We hypothesized that breast cancer cells must express brain-associated markers that would enable their invasion and survival in the brain microenvironment. We assessed a panel of brain-predominant markers and found an elevation of several neuronal markers (βIII-tubulin, Nestin, and AchE) in brain metastatic breast cancer cells. Among these neuronal predominant markers, in silico analysis revealed overexpression of βIII-tubulin (TUBB3) in breast cancer brain metastases (BCBM) and its expression was significantly associated with distant metastases. TUBB3 knockdown studies were conducted in breast cancer models (MDA-Br, GLIM2, and MDA-MB-468), which revealed significant reduction in their invasive capabilities. MDA-Br cells with suppressed TUBB3 also demonstrated loss of key signaling molecules such as β3 integrin, pFAK, and pSrc in vitro. Furthermore, TUBB3 knockdown in a brain metastatic breast cancer cell line compromised its metastatic ability in vivo, and significantly improved survival in a brain metastasis model. These results implicate a critical role of TUBB3 in conferring brain metastatic potential to breast cancer cells. Mol Cancer Ther; 14(5); 1152–61. ©2015 AACR.

Glioblastoma (GBM) is characterized by extremely poor prognoses, despite the use of gross surgical resection, alkylating chemotherapeutic agents, and radiotherapy. Evidence increasingly highlights the role of the tumor microenvironment in enabling this aggressive phenotype. Despite this interest, the role of neurotransmitters, brain-specific messengers underlying synaptic transmission, remains murky. These signaling molecules influence a complex network of molecular pathways and cellular behaviors in many CNS-resident cells, including neural stem cells and progenitor cells, neurons, and glia cells. Critically, available data convincingly demonstrate that neurotransmitters can influence proliferation, quiescence, and differentiation status of these cells. This ability to affect progenitors and glia-GBM-initiating cells-and their availability in the CNS strongly support the notion that neurotransmitters participate in the onset and progression of GBM. This review will focus on dopamine and serotonin, as studies indicate they contribute to gliomagenesis. Particular attention will be paid to how these neurotransmitters and their receptors can be utilized as novel therapeutic targets. Overall, this review will analyze the complex biology governing the interaction of GBM with neurotransmitter signaling and highlight how this interplay shapes the aggressive nature of GBM.

Few quantifiable tissue biomarkers for the diagnosis and prognosis of prostate cancer exist. Using an unbiased, quantitative approach, this study evaluates the potential of three proteins of the 40S ribosomal protein complex as putative biomarkers of malignancy in prostate cancer. Prostate tissue arrays, constructed from 82 patient samples (245 tissue cores, stage pT3a or pT3b), were stained for antibodies against three ribosomal proteins, RPS19, RPS21 and RPS24. Semi-automated Ox-DAB signal quantification using ImageJ software revealed a significant change in expression of RPS19, RPS21 and RPS24 in malignant vs non-malignant tissue (p

Brain tumor-initiating cells (BTICs) have been identified as key contributors to therapy resistance, recurrence, and progression of diffuse gliomas, particularly glioblastoma (GBM). BTICs are elusive therapeutic targets that reside across the blood-brain barrier, underscoring the urgent need to develop novel therapeutic strategies. Additionally, intratumoral heterogeneity and adaptations to therapeutic pressure by BTICs impede the discovery of effective anti-BTIC therapies and limit the efficacy of individual gene targeting. Recent discoveries in the genetic and epigenetic determinants of BTIC tumorigenesis offer novel opportunities for RNAi-mediated targeting of BTICs. Here we show that BTIC growth arrest in vitro and in vivo is accomplished via concurrent siRNA knockdown of four transcription factors (SOX2, OLIG2, SALL2, and POU3F2) that drive the proneural BTIC phenotype delivered by multiplexed siRNA encapsulation in the lipopolymeric nanoparticle 7C1. Importantly, we demonstrate that 7C1 nano-encapsulation of multiplexed RNAi is a viable BTIC-targeting strategy when delivered directly in vivo in an established mouse brain tumor. Therapeutic potential was most evident via a convection-enhanced delivery method, which shows significant extension of median survival in two patient-derived BTIC xenograft mouse models of GBM. Our study suggests that there is potential advantage in multiplexed targeting strategies for BTICs and establishes a flexible nonviral gene therapy platform with the capacity to channel multiplexed RNAi schemes to address the challenges posed by tumor heterogeneity.

A limitation of small molecule inhibitors, nanoparticles (NPs) and therapeutic adenoviruses is their incomplete distribution within the entirety of solid tumors such as malignant gliomas. Currently, cell-based carriers are making their way into the clinical setting as they offer the potential to selectively deliver many types of therapies to cancer cells.Here, we review the properties of stem cells, induced pluripotent stem cells and engineered cells that possess the tumor-tropic behavior necessary to serve as cell carriers. We also report on the different types of therapeutic agents that have been delivered to tumors by these cell carriers, including: i) therapeutic genes; ii) oncolytic viruses; iii) NPs; and iv) antibodies. The current challenges and future promises of cell-based drug delivery are also discussed.While the emergence of stem cell-mediated therapy has resulted in promising preclinical results and a human clinical trial utilizing this approach is currently underway, there is still a need to optimize these delivery platforms. By improving the loading of therapeutic agents into stem cells and enhancing their migratory ability and persistence, significant improvements in targeted cancer therapy may be achieved.

In 1908, Oluf Bang and Vilhelm Ellerman laid the foundation for theory of oncoviruses by demonstrating that the avian erythroblastosis (a form of chicken leukaemia) could be transmitted by cell-free extracts. Since then, it has been shown very convincingly that viruses can directly cause several human cancers by various mechanisms. Epidemiological data imply that viruses are the second most important risk factor for cancer development in humans, exceeded only by tobacco consumption. Although the ability of certain viruses (hepatitis B and C, human papillomavirus, etc) to cause cancer has been time tested and proven scientifically, there are several other potential viral candidates whose role in oncogenesis is more controversial. One such controversial scenario involves the role of cytomegalovirus (CMV) in malignant gliomas, the most common form of primary brain tumour. CMV first attracted attention about a decade ago when CMV gene products were found in glioma tissue but not in normal brain. Since this initial observation, several different groups have shown an oncomodulatory effect of CMV; however, direct association between CMV infection and incidence of glioma is lacking. In this review, we will evaluate the evidence, both preclinical and clinical, regarding the possible role of CMV in gliomagenesis and maintenance. We will also critically evaluate the rationale for using antiviral drugs in the treatment of patients with glioma.

There will be an estimated 1.8 million new cancer diagnoses in the US this year alone, with >80% of these cases driven by oncogenic K-RAS/SIAH pathway activation, even in the absence of oncogenic RAS mutations. Over three decades of intense research has focused on conquering RAS pathway activation in cancer, yet oncogenic K-RAS has remained essentially undruggable. Seven-IN-Absentia (SINA) is an evolutionarily conserved E3 ubiquitin ligase that is the most downstream signaling module identified in the RAS signaling pathway thus far. Underscoring SINA’s importance is its high degree of evolutionary conservation with over 83% amino acid identity shared between Drosophila SINA and its human SINA homologs (SIAHs). As a major signaling “gatekeeper” in the RAS pathway, we have shown that SIAH is required for oncogenic K-RAS-driven tumorigenesis and metastasis in human pancreatic, lung, and breast cancers. Since SIAHs appear to be the ideal drug target to inhibit “undruggable” K-RAS activation, it is important to precisely characterize the activity, regulation, and substrate targeting mechanism(s) of this highly conserved family of SINA/SIAH E3 ligases. By deploying the Drosophila photoreceptor development system, we conducted a genetic modifier screen and identified 28 new sina mutant alleles that exhibit a range of mutant phenotypes. For example, sina complete loss of function (null alleles) is cell lethal, and several newly identified point mutant alleles exhibit stronger mutant phenotypes than those of previously published truncated alleles. Sequencing analysis of these sinamutant revealed critical roles of several immutable amino acid residues indispensable for SINA function. To demonstrate the functional conservation of SINA/SIAH proteins, we generated a collection of transgenic fly lines that carry either wild-type (WT) or dominant negative (DN) DmSINA and human SIAH1/2. Tissue-specific SINA/SIAH expression using GAL4 drivers revealed the biological consequences of gain or loss of function of SINA/SIAH1/2 in transmitting RAS signaling in Drosophila development. Immunofluorescent (IF) staining of developing imaginal discs and electron micrographs of adult tissues show that ectopic expression of SINAWT/DN/SIAHWT/DN in neurons resulted dramatic changes in neuronal cell fate in the developing eye and sensory neuronal cells on the notum. Furthermore, IF staining of larval salivary glands revealed a critical role for SINA/SIAH in regulating cell shape, focal adhesions, and cellular junctions. Results from these transgenic models suggest that Drosophila SINA and human SIAH1/2 are highly evolutionarily conserved and functionally interchangeable. Ultimately, we aim to translate these preclinical findings to demonstrate the high potency and anticancer efficacy of an anti-SIAH-based anti-K-RAS strategy against multidrug-resistant and incurable human cancer. Citation Format: Robert E. Van Sciver, Yajun Cao, Atique U. Ahmed, Amy H. Tang. The critical role of seven-in-absentia (SINA) family E3 ligases in normal development and oncogenic K-RAS-driven human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 83.

Current research has evaluated the intrinsic tumor-tropic properties of stem cell carriers for targeted anticancer therapy. Our laboratory has been extensively studying in the preclinical setting, the role of neural stem cells (NSCs) as delivery vehicles of CRAd-S-pk7, a gliomatropic oncolytic adenovirus (OV). However, the mediated toxicity of therapeutic payloads, such as oncolytic adenoviruses, toward cell carriers has significantly limited this targeted delivery approach. Following this rationale, in this study, we assessed the role of a novel antioxidant thiol, N-acetylcysteine amide (NACA), to prevent OV-mediated toxicity toward NSC carriers in an orthotropic glioma xenograft mouse model. Our results show that the combination of NACA and CRAd-S-pk7 not only increases the viability of these cell carriers by preventing reactive oxygen species (ROS)-induced apoptosis of NSCs, but also improves the production of viral progeny in HB1.F3.CD NSCs. In an intracranial xenograft mouse model, the combination treatment of NACA and NSCs loaded with CRAd-S-pk7 showed enhanced CRAd-S-pk7 production and distribution in malignant tissues, which improves the therapeutic efficacy of NSC-based targeted antiglioma oncolytic virotherapy. These data demonstrate that the combination of NACA and NSCs loaded with CRAd-S-pk7 may be a desirable strategy to improve the therapeutic efficacy of antiglioma oncolytic virotherapy.

Glioblastoma multiforme (GBM) remains fatal despite intensive surgical, radiotherapeutic, and chemotherapeutic interventions. Neural stem cells (NSCs) have been used as cellular vehicles for the transportation of oncolytic virus (OV) to therapeutically resistant and infiltrative tumor burdens throughout the brain. The HB1.F3-CD human NSC line has demonstrated efficacy as a cell carrier for the delivery of a glioma tropic OV CRAd-Survivin-pk7 (CRAd-S-pk7) in vitro and in animal models of glioma. At this juncture, no study has investigated the effectiveness of OV-loaded NSCs when applied in conjunction with the standard of care for GBM treatment, and therefore this study was designed to fill this void. Here, we show that CRAd-S-pk7-loaded HB1.F3-CD cells retain their tumor-tropic properties and capacity to function as in situ viral manufacturers in the presence of ionizing radiation (XRT) and temozolomide (TMZ). Furthermore, for the first time, we establish a logical experimental model that aims to recapitulate the complex clinical scenario for the treatment of GBM and tests the compatibility of NSCs loaded with OV. We report that applying OV-loaded NSCs together with XRT and TMZ can increase the median survival of glioma bearing mice by approximately 46%. Most importantly, the timing and order of therapeutic implementation impact therapeutic outcome. When OV-loaded NSCs are delivered prior to rather than after XRT and TMZ treatment, the median survival of mice bearing patient-derived GBM43 glioma xenografts is extended by 30%. Together, data from this report support the testing of CRAd-S-pk7-loaded HB1.F3-CD cells in the clinical setting and argue in favor of a multimodality approach for the treatment of patients with GBM.

A 3-step glioblastoma-tropic delivery and therapy method using nanoparticle programmed self-destructive neural stem cells (NSCs) is demonstrated in vivo: 1) FDA-approved NSCs for clinical trials are loaded with pH-sensitive MSN-Dox; 2) the nanoparticle conjugates provide a delayed drug-releasing mechanism and allow for NSC migration towards a distant tumor site; 3) NSCs eventually undergo cell death and release impregnated MSN-Dox, which subsequently induces toxicity towards surrounding glioma cells.

Neural stem cells (NSCs) are defined as multipotent progenitor cells that originate from the developing and adult central nervous system (1). NSCs display intrinsic tumor tropism that can be exploited for targeted anticancer drug delivery to invasive and metastatic cancer (2,3). In theory, the tumor-homing property of NSCs offers a substantial advantage over other targeted therapies, such as antibody-directed drug delivery, because of their ability to detect various cues generated by satellite tumor foci and respond to such cues by extravasating through complex tissue microenvironments and migrating to distant diseased areas (4). Glioblastoma multiforme (GBM) is the most common and aggressive primary central nervous system tumor in adults and is characterized by its propensity to infiltrate throughout the brain and cause relapses in patients because of the existence of an aberrant chemo- and radio-resistant glioma stem cell (GSC) population (5). Thus, a true cure for this formidable disease cannot arise from the application of traditional antineoplastic principles; it requires a dynamic agent capable of targeting scattered disease lesions as well as eliminating the tumor-initiating cancer stem cells effectively with minimal disruption of the existing delicate neural architecture (6). Based on this, our lab has extensively evaluated NSCs as a cellular vehicle for the targeted delivery of glioma tropic oncolytic adenoviral virotherapy (OV) CRAd-S-pk7 (7–9). We have proposed that by combining NSCs’ unique tumor tropism with the OV’s ability to target chemo- and radio-resistant GSCs (6,10) we may overcome the deficiencies inherent to each approach deployed in isolation and can effectively target GBM. As a proof-of-principle we have previously demonstrated that 1) NSCs can be used as cellular vehicles for the in vivo delivery of an OV to intracranial gliomas (7); 2) intratumoral delivery of NSCs loaded with the CRAd-S-pk7, a glioma-tropic OV regulated by the tumor-specific survivin promoter (7,11), increased median survival by 50% compared with mice treated with OV alone in an orthotopic xenograft model of human glioma (8); and 3) NSCs demonstrated superior therapeutic efficacy when compared with mesenchymal stem cells as a cell carrier for OV in the context of intracranial gliomas (9). Because these previously published results argue in favor of using NSCs as targeted cellular delivery vehicles for antiglioma oncolytic virotherapy, we conducted the following critical translational studies to justify its application in a phase I clinical trial for patients with GBM: 1) identified an optimal NSC-based cell carrier for antiglioma oncolytic virotherapy; 2) tested the selected NSC-based cell carrier in several diverse and clinically relevant glioma xenograft models; 3) developed a noninvasive imaging protocol to monitor in vivo distribution and migratory activity of NSC-based cell carriers in real time; 4) examined the capacity for the NSC-based cell carrier to deliver antiglioma OV to a distant tumor burden in a glioma xenograft model; and 5) evaluated the therapeutic efficacy of NSC-based oncolytic virotherapy in a distance-delivery glioma xenograft model. In this report, we provide a detailed evaluation of two immortalized NSC lines as cell carriers for targeted antiglioma therapy. Our results indicate that HB1.F3.CD, a US Food and Drug Administration–approved NSC line for human clinical trials (NCT01172964) is the most suitable NSC cell carrier for the future application of cell-based OV delivery in the clinical setting. We found that HB1.F3.CD cells can effectively hand off the viral therapeutic payload to distant tumor sites and substantially prolong median survival in diverse orthotopic models of human glioma. Thus, data presented in this study solidify the notion that NSCs can be used as cell carriers for the targeted delivery of antiglioma oncolytic viruses and serve as the foundation for an investigational new drug application for a human clinical trial involving newly diagnosed and recurrent patients with malignant gliomas.

Glioblastoma multiforme (GBM), the most common and aggressive form of primary brain tumor, presents a dismal prognosis. Current standard therapies are only able to improve patient survival by a few months. The search for alternative approaches in glioblastoma treatment, together with the recent discovery of a new class of small RNA molecules that are capable of regulating gene expression, prompted a race for a deeper and thorough understanding of how these molecules work. Today, it is known that microRNAs are involved in many cellular processes that are altered in GBM tumors, such as angiogenesis, invasion, cell proliferation and apoptosis. Research in this area is now gathering efforts to translate these findings into clinically relevant therapies that could improve the diagnosis and outcome of GBM patients. In this review, we discuss the use of microRNAs as potential diagnostic, prognostic and therapeutic tools against glioblastoma. We will also assess the current challenges and future perspectives of microRNA-based therapies, with a special focus on why this promising therapeutic approach is not yet in the clinic and how to overcome this limitation.

// Brenda Auffinger 1 , Ramin Morshed 1 , Alex Tobias 1 , Yu Cheng 1 , Atique U Ahmed 1 , Maciej S Lesniak 1 1 The Brain Tumor Center, The University of Chicago, Chicago, Illinois, USA Correspondence: Maciej S Lesniak, email: // Keywords : Nanoparticle, drug delivery systems, stem cell carriers, malignant glioma, brain cancer, targeted delivery. Received : March 15, 2013 Accepted : March 24, 2013 Published : March 26, 2013 Abstract Despite all recent advances in malignant glioma research, only modest progress has been achieved in improving patient prognosis and quality of life. Such a clinical scenario underscores the importance of investing in new therapeutic approaches that, when combined with conventional therapies, are able to effectively eradicate glioma infiltration and target distant tumor foci. Nanoparticle-loaded delivery systems have recently arisen as an exciting alternative to improve targeted anti-glioma drug delivery. As drug carriers, they are able to efficiently protect the therapeutic agent and allow for sustained drug release. In addition, their surface can be easily manipulated with the addition of special ligands, which are responsible for enhancing tumor-specific nanoparticle permeability. However, their inefficient intratumoral distribution and failure to target disseminated tumor burden still pose a big challenge for their implementation as a therapeutic option in the clinical setting. Stem cell-based delivery of drug-loaded nanoparticles offers an interesting option to overcome such issues. Their ability to incorporate nanoparticles and migrate throughout interstitial barriers, together with their inherent tumor-tropic properties and synergistic anti-tumor effects make these stem cell carriers a good fit for such combined therapy. In this review, we will describe the main nanoparticle delivery systems that are presently available in preclinical and clinical studies. We will discuss their mechanisms of targeting, current delivery methods, attractive features and pitfalls. We will also debate the potential applications of stem cell carriers loaded with therapeutic nanoparticles in anticancer therapy and why such an attractive combined approach has not yet reached clinical trials.

Cancer handles an estimated 7.6 million deaths worldwide per annum. A recent theory focuses on the role Cancer Stem Cells (CSCs) in driving tumorigenesis and disease progression. This theory hypothesizes that a population of the tumor cell with similar functional and phenotypic characteristics as normal tissue stem cells are responsible for formation and advancement of many human cancers. The CSCs subpopulation can differentiate into non-CSC tumor cells and promote phenotypic and functional heterogeneity within the tumor. The presence of CSCs has been reported in a number of human cancers including blood, breast, brain, colon, lung, pancreas prostate and liver. Although the origin of CSCs remains a mystery, recent reports suggest that the phenotypic characteristics of CSCs may be plastic and are influenced by the microenvironment specific for the individual tumor. Such factors unique to each tumor preserve the dynamic balance between CSCs to non-CSCs cell fate, as well as maintain the proper equilibrium. Alternating such equilibrium via dedifferentiation can result in aggressiveness, as CSCs are considered to be more resistant to the conventional cancer treatments of chemotherapy and radiation. Understanding how the tumoral microenvironment affects the plasticity driven CSC niche will be critical for developing a more effective treatment for cancer by eliminating its aggressive and recurring nature that now is believed to be perpetuated by CSCs.

Background CRAd-S-pk7 is a conditionally replicative oncolytic adenoviral vector that contains a survivin promoter and a pk7 fiber modification that confer tumor-specific transcriptional targeting and preferential replication in glioma while sparing the surrounding normal brain parenchyma. Methods This IND-enabling study performed under GLP conditions evaluated the toxicity and biodistribution of CRAd-S-pk7 administered as a single intracerebral dose to Syrian hamsters, a permissive model of adenoviral replication. Two hundred and forty animals were stereotactically administered either vehicle (n = 60) or CRAd-S-pk7 at 2.5 × 107, 2.5 × 108, or 2.5 × 109 viral particles (vp)/animal (each n = 60) on day 1. The animals were closely monitored for toxicology evaluation, assessment of viral distribution, and immunogenicity of CRAd-S-pk7. Results Changes in hematology, clinical chemistry, and coagulation parameters were minor and transient, and consistent with the inflammatory changes observed microscopically. These changes were considered to be of little toxicological significance. The vector remained localized primarily in the brain and to some degree in the tissues at the incision site. Low levels of vector DNA were detected in other tissues in a few animals suggesting systemic circulation of the virus. Viral DNA was detected in brains of hamsters for up to 62 days. However, microscopic changes and virus-related toxicity to the central nervous system were considered minor and decreased in incidence and severity over time. Such changes are not uncommon in studies using adenoviral vectors. Conclusion This study provides safety and toxicology data justifying a clinical trial of CRAd-S-pk7 loaded in FDA-approved HB1.F3.CD neural stem cell carriers administered at the tumor resection bed in humans with recurrent malignant glioma. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0895-8) contains supplementary material, which is available to authorized users.

With the median survival of 14.6 months following best available standard of care, malignant gliomas (MGs) remain one of the biggest therapeutic challenges of the modern time. Although the last several decades have witnessed tremendous advancement in our understanding of MG and evolution of many successful preclinical therapeutic strategies, even the most successful preclinical therapeutic strategies often fail to cross the phase I/II clinical trial threshold. One of the significant, but less commonly discussed, barriers in developing effective glioma therapy is the lack of a robust preclinical model. For the last 30 years, rodent orthotopic xenograft models have been extensively used in the preclinical setting. Although they provide a good basic model for understanding tumor biology, their value in successfully translating preclinical therapeutic triumph into clinical success is extremely poor. Companion dogs, which share the same environmental stress as their human counterparts, also spontaneously develop MGs. Dog gliomas that develop spontaneously in an immunocompetent host are very similar to human gliomas and potentially provide a stronger platform for validating the efficacy of therapeutic strategies proven successful in preclinical mouse models. Integrating this model can accelerate development of effective therapeutic options that will benefit both human subjects and pet dogs.

Glioblastoma multiforme (GBM) is a highly invasive brain tumour that is unvaryingly fatal in humans despite even aggressive therapeutic approaches such as surgical resection followed by chemotherapy and radiotherapy. Unconventional treatment options such as gene therapy provide an intriguing option for curbing glioma related deaths. To date, gene therapy has yielded encouraging results in preclinical animal models as well as promising safety profiles in phase I clinical trials, but has failed to demonstrate significant therapeutic efficacy in phase III clinical trials. The most widely studied antiglioma gene therapy strategies are suicide gene therapy, genetic immunotherapy and oncolytic virotherapy, and we have attributed the challenging transition of these modalities into the clinic to four major roadblocks: (1) anatomical features of the central nervous system, (2) the host immune system, (3) heterogeneity and invasiveness of GBM and (4) limitations in current GBM animal models. In this review, we discuss possible ways to jump these hurdles and develop new gene therapies that may be used alone or in synergy with other modalities to provide a powerful treatment option for patients with GBM.

Conditionally replicating adenoviruses (CRAd) are a promising class of gene therapy agents that can overcome already known glioblastoma (GBM) resistance mechanisms but have limited distribution upon direct intratumoral (i.t.) injection. Collagen bundles in the extracellular matrix (ECM) play an important role in inhibiting virus distribution. In fact, ECM pre-treatment with collagenases improves virus distributions to tumor cells. Matrix metalloproteinases (MMPs) are an endogenous class of collagenases secreted by tumor cells whose function can be altered by different drugs including anti-angiogenic agents, such as bevacizumab. In this study we hypothesized that up-regulation of MMP activity during antiangiogenic therapy can improve CRAd-S-pk7 distribution in GBM. We find that MMP-2 activity in human U251 GBM xenografts increases (*p=0.03) and collagen IV content decreases (*p=0.01) during vascular endothelial growth factor (VEGF-A) antibody neutralization. After proving that collagen IV inhibits CRAd-S-pk7 distribution in U251 xenografts (Spearman rho= −0.38; **p=0.003), we show that VEGF blocking antibody treatment followed by CRAd-S-pk7 i.t. injection reduces U251 tumor growth more than each individual agent alone (***p