TMOD-03. A SWINE MODEL OF GLIOBLASTOMA INDUCED BY SOMATIC GENE MODIFICATION

Abstract Glioblastoma (GBM) is the most common and malignant primary brain tumor. Novel therapeutic development for GBM is desperately needed, as the standard of care universally fails to cure patients and the 5-year survival rate remains extremely low. GBM therapeutic development is hampered by the lack of relevant preclinical models for preclinical studies. To mitigate this problem, we have developed a genetic model of GBM in outbred, immune-proficient swine which have comparable brain size and anatomy to humans. We developed methods for introducing genome engineering tools to minipig brain in vivo by direct injection of gene delivery reagents to the lateral ventricle, altering major signaling pathways frequently changed in human GBM. Using this technique, we have delivered a combination of expression vectors for oncogenes and targeted nucleases to disrupt tumor suppressor genes commonly altered in human GBM. We have altered six major human GBM-associated signaling pathways and modeled molecular GBM subclasses. We have also engineered a secreted tumor reporter that can be used to monitor tumor size through a simple blood test. This somatic cell gene-modification platform we have developed in the minipig allows us to reproduce the genetic heterogeneity seen in GBM and understand the impact of the tumor microenvironment, immune system, and response to therapy. This minipig model of GBM Is being used to test the standard of care against novel therapies in preclinical studies, and biopsy, surgical, imaging, and radiation therapy techniques are being optimized in this mode to improve clinical trial success rates and patient outcomes. Funding for this study is provided by the National Institutes of Health though SBIR grant # 1R43CA235837-01A1.

Download Full-text

TMOD-20. A SWINE MODEL OF GLIOBLASTOMA INDUCED BY SOMATIC GENE MODIFICATION

Neuro-Oncology ◽

10.1093/neuonc/noaa215.971 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii232-ii232

Author(s):

Barbara Tschida ◽

Dylan Duerre ◽

Mandy Taisto ◽

Adrienne Watson

Keyword(s):

Brain Size ◽

Expression Vectors ◽

Efficient Production ◽

Swine Model ◽

Large Animal ◽

Gene Modification ◽

Large Animal Models ◽

Therapeutic Development ◽

Human Gbm

Abstract Glioblastoma (GBM) is the most common and malignant primary brain tumor. Novel therapeutic development for GBM is needed since the standard of care universally fails to cure patients and the five-year survival rate remains below 10%. GBM therapeutic development is hampered by the lack of relevant large animal models for preclinical studies. To mitigate this problem, we are developing a model of GBM in outbred, immune-proficient swine which have comparable brain size and anatomy to humans. We developed methods for introducing genome engineering tools to minipig brain cells in vivo by direct injection of gene delivery reagents to the lateral ventricle. Using this technique, we have delivered a combination of expression vectors for oncogenes and targeted nucleases to disrupt tumor suppressor genes commonly altered in human GBM to alter six major human GBM-associated signaling pathways in a cohort of minipigs (Ras, Pi3k, p53, Rb/E2F, Pdgf, and the alternative lengthening of telomeres (ALT) pathways). These minipigs are being monitored for tumorigenesis using a secreted reporter, detectable through a simple luminescence-based blood test. Resulting tumors will be examined molecularly to detect the pathway-associated alterations in tumor tissue and determine the resemblance to human GBM. We hypothesize that this somatic cell gene-modification platform we have developed in the minipig will facilitate the efficient production of brain tumors that histologically and genetically resemble human GBM. It will allow the production of tumors that are genetically heterogeneous, of specified molecular subclasses, containing therapeutic targets of interest, and in the context of genetic backgrounds of interest. This minipig model of GBM will be applied towards preclinical therapeutic studies, imaging studies using human clinical grade equipment, and surgical technique development, to improve clinical trial success rates and patient outcomes. Funding for this study is provided by the National Institutes of Health through SBIR grant # 1R43CA235837-01A1.

Download Full-text

TMOD-17. A SWINE MODEL OF GLIOBLASTOMA INDUCED BY SOMATIC GENE MODIFICATION

Neuro-Oncology ◽

10.1093/neuonc/noz175.1116 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi266-vi266

Author(s):

Barbara Tschida ◽

Dylan Duerre ◽

Mandy Taisto ◽

Staci Solin ◽

Adrienne Watson

Keyword(s):

Signaling Pathways ◽

Gene Editing ◽

Brain Size ◽

Rapid Development ◽

Swine Model ◽

Large Animal ◽

Induced Mutations ◽

Gene Modification ◽

Large Animal Models ◽

Human Gbm

Abstract Glioblastoma (GBM) is the most common and malignant primary brain tumor. New, effective treatments for GBM are needed since the standard of care universally fails to cure patients. A major hurdle for GBM therapeutic development is the lack of relevant large animal models with high translational value for preclinical studies. To overcome this, we are developing a model of GBM in outbred, immune-proficient swine which have comparable brain size and anatomy to humans. GBM tumors will be driven by somatic alterations to major signaling pathways frequently altered in human GBM and will express a secreted reporter for tumor growth detectable in the peripheral blood. These alterations and reporter expression cassettes will be induced by delivering transposons and CRISPR/Cas9 gene-editing tools to the subventricular brain cells of live swine by stereotactic injections. We have developed and deployed these gene-editing tools, demonstrating their efficacy in swine cells. We have identified stereotactic coordinates to reproducibly target the lateral ventricles of neonatal swine brains and have demonstrated successful plasmid delivery to cells at these coordinates. Stable, persistent transposon integration and clonal expansion of modified cells has been confirmed by sequencing transposon-genomic DNA junctions in brain tissue six-months post-injection. To induce GBM-like tumors, we have introduced gene delivery and gene editing reagents to alter six major human GBM-associated signaling pathways in a cohort of swine. Resulting tumors will be examined molecularly to detect the pathway-associated transposons and CRISPR/Cas9-induced mutations in tumor tissue and determine the resemblance to human GBM. This somatic cell gene-modification platform will be adaptable, allowing on-demand inclusion of preclinical study-relevant alterations in GBM tumors, and will allow the rapid development tumors histologically and genetically similar to human GBM, which will be valuable for use in pre-clinical therapeutic studies, imaging studies using human clinical grade equipment, and surgical technique development.

Download Full-text

The evolution of alternative splicing in glioblastoma under therapy

Genome Biology ◽

10.1186/s13059-021-02259-5 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Lin Wang ◽

Karin Shamardani ◽

Husam Babikir ◽

Francisca Catalan ◽

Takahide Nejo ◽

...

Keyword(s):

Alternative Splicing ◽

Standard Of Care ◽

Surface Proteins ◽

Adult Brain ◽

Primary Tumors ◽

Cell Therapies ◽

Therapeutic Development ◽

Human Gbm ◽

Tissue Specimens ◽

Splicing Patterns

Abstract Background Alternative splicing is a rich source of tumor-specific neoantigen targets for immunotherapy. This holds promise for glioblastomas (GBMs), the most common primary tumors of the adult brain, which are resistant to standard-of-care therapy. Although most clinical trials enroll patients at recurrence, most preclinical studies have been done with specimens from primary disease. There are limited expression data from GBMs at recurrence and surprisingly little is known about the evolution of splicing patterns under therapy. Result We profile 37 primary-recurrent paired human GBM specimens via RNA sequencing. We describe the landscape of alternative splicing in GBM at recurrence and contrast that to primary and non-malignant brain-tissue specimens. By screening single-cell atlases, we identify cell-type-specific splicing patterns and novel splicing events in cell-surface proteins that are suitable targets for engineered T cell therapies. We identify recurrent-specific isoforms of mitogen-activated kinase pathway genes that enhance invasiveness and are preferentially expressed by stem-like cells. Conclusion These studies shed light on gene expression in recurrent GBM and identify novel targets for therapeutic development.

Download Full-text

Preclinical models in the study of sex differences

Clinical Science ◽

10.1042/cs20160847 ◽

2017 ◽

Vol 131 (6) ◽

pp. 449-469 ◽

Cited By ~ 10

Author(s):

Maria Buoncervello ◽

Matteo Marconi ◽

Alessandra Carè ◽

Paola Piscopo ◽

Walter Malorni ◽

...

Keyword(s):

Sex Differences ◽

State Of The Art ◽

Great Majority ◽

Response To Therapy ◽

Preclinical Studies ◽

Biological Mechanisms ◽

Preclinical Models ◽

The Impact

The biology of sex differences deals with the study of the disparities between females and males and the related biological mechanisms. Gender medicine focuses on the impact of gender and sex on human physiology, pathophysiology and clinical features of diseases that are common to women and men. The term gender refers to a complex interrelation and integration of sex–as a biological and functional determinant–and psychological and cultural behaviours (due to ethnical, social or religious background). The attention to the impact of gender differences on the pathophysiology and, therefore, on the clinical management of the most common diseases, such as cardiovascular diseases (CVD), neurodegenerative disorders, immune and autoimmune diseases as well as several tumours, is in fact often neglected. Hence, studies covering different fields of investigation and including sex differences in the pathogenesis, in diagnostic and prognostic criteria as well as in response to therapy appear mandatory. However, prerequisites for this development are preclinical studies, including in vitro and in vivo approaches. They represent the first step in the development of a drug or in the comprehension of the pathogenetic mechanisms of diseases, in turn a necessary step for the development of new or more appropriate therapeutic strategies. However, sex differences are still poorly considered and the great majority of preclinical studies do not take into account the relevance of such disparities. In this review, we describe the state of the art of these studies and provide some paradigmatic examples of key fields of investigation, such as oncology, neurology and CVD, where preclinical models should be improved.

Download Full-text

The Impact of Royal Jelly against Hepatic Ischemia/Reperfusion-Induced Hepatocyte Damage in Rats: The Role of Cytoglobin, Nrf-2/HO-1/COX-4, and P38-MAPK/NF-κB-p65/TNF-α Signaling Pathways

Current Molecular Pharmacology ◽

10.2174/1874467213666200514223829 ◽

2020 ◽

Vol 14 (1) ◽

pp. 88-100

Author(s):

Fares E.M. Ali ◽

Heba M. Saad Eldien ◽

Nashwa A.M. Mostafa ◽

Abdulrahman H. Almaeen ◽

Mohamed R.A. Marzouk ◽

...

Keyword(s):

Signaling Pathways ◽

P38 Mapk ◽

Royal Jelly ◽

Ischemia Reperfusion ◽

Histopathological Changes ◽

Down Regulation ◽

Hepatic Ischemia ◽

Tnf Α ◽

Hepatic Ischemia Reperfusion ◽

The Impact

Objective: The present study was conducted to elucidate the underlying molecular mechanism as well as the potential hepatoprotective effects of royal jelly (RJ) against hepatic ischemia/reperfusion (IR) injury. Methods: Rats were assigned into four groups; sham (received vehicle), IR (30 minutes ischemia and 45 minutes reperfusion), sham pretreated with RJ (200 mg/kg P.O.), and IR pretreated with RJ (200 mg/kg P.O.). The experiment has lasted for 28 days. Results: Hepatic IR significantly induced hepatic dysfunctions, as manifested by elevation of serum transaminases, ALP and LDH levels. Moreover, hepatic IR caused a significant up-regulation of P38-MAPK, NF-κB-p65, TNF-α and MDA levels along with marked down-regulation of Nrf-2, HO-1, COX-4, cytoglobin, IκBa, IL-10, GSH, GST and SOD levels. Additionally, marked histopathological changes were observed after hepatic IR injury. On the contrary, pretreatment with RJ significantly improved hepatic functions along with the alleviation of histopathological changes. Moreover, RJ restored oxidant/antioxidant balance as well as hepatic expressions of Nrf-2, HO-1, COX-4, and cytoglobin. Simultaneously, RJ significantly mitigated the inflammatory response by down-regulation of P38-MAPK, NF-κB-p65, TNF-α expression. Conclusion: The present results revealed that RJ has successfully protected the liver against hepatic IR injury through modulation of cytoglobin, Nrf-2/HO-1/COX-4, and P38-MAPK/NF-κB-p65/TNF-α signaling pathways.

Download Full-text

Updated Insights on EGFR Signaling Pathways in Glioma

International Journal of Molecular Sciences ◽

10.3390/ijms22020587 ◽

2021 ◽

Vol 22 (2) ◽

pp. 587

Author(s):

Alexandru Oprita ◽

Stefania-Carina Baloi ◽

Georgiana-Adeline Staicu ◽

Oana Alexandru ◽

Daniela Elise Tache ◽

...

Keyword(s):

Signaling Pathways ◽

Treatment Strategies ◽

Growth Factor Receptor ◽

Standard Of Care ◽

Egfr Signaling ◽

Current Standard ◽

Egfr Amplification ◽

Epidermal Growth ◽

New Diagnosis ◽

Clear Clinical Benefit

Nowadays, due to recent advances in molecular biology, the pathogenesis of glioblastoma is better understood. For the newly diagnosed, the current standard of care is represented by resection followed by radiotherapy and temozolomide administration, but because median overall survival remains poor, new diagnosis and treatment strategies are needed. Due to the quick progression, even with aggressive multimodal treatment, glioblastoma remains almost incurable. It is known that epidermal growth factor receptor (EGFR) amplification is a characteristic of the classical subtype of glioma. However, targeted therapies against this type of receptor have not yet shown a clear clinical benefit. Many factors contribute to resistance, such as ineffective blood–brain barrier penetration, heterogeneity, mutations, as well as compensatory signaling pathways. A better understanding of the EGFR signaling network, and its interrelations with other pathways, are essential to clarify the mechanisms of resistance and create better therapeutic agents.

Download Full-text

Inflammation and tumor progression: signaling pathways and targeted intervention

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-021-00658-5 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Huakan Zhao ◽

Lei Wu ◽

Guifang Yan ◽

Yu Chen ◽

Mingyue Zhou ◽

...

Keyword(s):

Growth Factor ◽

Tumor Progression ◽

Signaling Pathways ◽

Transforming Growth Factor ◽

Response To Therapy ◽

Janus Kinase ◽

Oncolytic Viruses ◽

Mitogen Activated Protein Kinase ◽

Resolution Of Inflammation ◽

Chemokine Ligands

AbstractCancer development and its response to therapy are regulated by inflammation, which either promotes or suppresses tumor progression, potentially displaying opposing effects on therapeutic outcomes. Chronic inflammation facilitates tumor progression and treatment resistance, whereas induction of acute inflammatory reactions often stimulates the maturation of dendritic cells (DCs) and antigen presentation, leading to anti-tumor immune responses. In addition, multiple signaling pathways, such as nuclear factor kappa B (NF-kB), Janus kinase/signal transducers and activators of transcription (JAK-STAT), toll-like receptor (TLR) pathways, cGAS/STING, and mitogen-activated protein kinase (MAPK); inflammatory factors, including cytokines (e.g., interleukin (IL), interferon (IFN), and tumor necrosis factor (TNF)-α), chemokines (e.g., C-C motif chemokine ligands (CCLs) and C-X-C motif chemokine ligands (CXCLs)), growth factors (e.g., vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β), and inflammasome; as well as inflammatory metabolites including prostaglandins, leukotrienes, thromboxane, and specialized proresolving mediators (SPM), have been identified as pivotal regulators of the initiation and resolution of inflammation. Nowadays, local irradiation, recombinant cytokines, neutralizing antibodies, small-molecule inhibitors, DC vaccines, oncolytic viruses, TLR agonists, and SPM have been developed to specifically modulate inflammation in cancer therapy, with some of these factors already undergoing clinical trials. Herein, we discuss the initiation and resolution of inflammation, the crosstalk between tumor development and inflammatory processes. We also highlight potential targets for harnessing inflammation in the treatment of cancer.

Download Full-text

Impact of simple, specific, verbal instructions on the quality of bowel preparation in hospitalized patients undergoing colonoscopy: a multicenter randomized controlled trial

Endoscopy International Open ◽

10.1055/a-1339-0913 ◽

2021 ◽

Vol 09 (03) ◽

pp. E378-E387

Author(s):

Konstantinos Triantafyllou ◽

Paraskevas Gkolfakis ◽

Alexandros Skamnelos ◽

Georgia Diamantopoulou ◽

Athanasios Dagas ◽

...

Keyword(s):

Bowel Preparation ◽

Controlled Trial ◽

Statistical Significance ◽

Intervention Group ◽

Standard Of Care ◽

Hospitalized Patients ◽

Verbal Instructions ◽

Multicenter Randomized Controlled Trial ◽

The Impact

Abstract Background and study aims Bowel preparation for colonoscopy is frequently inadequate in hospitalized patients. We explored the impact of specific verbal instructions on the quality of inpatients bowel preparation and factors associated with preparation failure. Patients and methods Randomized (1:1), two strata (mobilized vs. bedridden; 3:2) trial of consecutive inpatients from four tertiary centers, who received either specific, verbal instructions or the standard of care (SOC) ward instructions about bowel preparation. The rate of adequate bowel preparation (Boston Bowel Preparation Score [BBPS] ≥ 6, no segment < 2) comprised the primary endpoint. Mean BBPS score, good (BBPS score ≥ 7, no segment score < 2) and excellent (BBPS = 9) were among secondary endpoints. Results We randomized 300 inpatients (180 mobile) aged 71.7 ± 15.1 years in the intervention (49.7 %) and SOC (50.3 %) groups, respectively. Overall, more patients in the intervention group achieved adequate bowel preparation, but this difference did not reach statistical significance neither in the intention-to-treat [90/149 (60.4 %) vs. 82/151 (54.3 %); P = 0.29] nor in the per-protocol analysis [90/129 (69.8 %) vs. 82/132 (62.1 %); P = 0.19]. Overall BBPS score did not differ statistical significantly in the two groups, but the provision of specific verbal instructions was associated with significant higher rates of good (58.1 % vs. 43.2 %; P = 0.02) and excellent (31.8 % vs. 16.7 %; P = 0.004) bowel preparation compared to the SOC group. Administration of same-day bowel preparation and patient American Society of Anesthesiologists score > 2 were identified as risk factors for inadequate bowel preparation. Conclusions Provision of specific verbal instructions did not increase the rate of adequate bowel preparation in a population of mobilized and bedridden hospitalized patients.

Download Full-text

Beyond Microsatellite Instability: Evolving Strategies Integrating Immunotherapy for Microsatellite Stable Colorectal Cancer

Current Treatment Options in Oncology ◽

10.1007/s11864-021-00870-z ◽

2021 ◽

Vol 22 (8) ◽

Author(s):

Federica Pecci ◽

Luca Cantini ◽

Alessandro Bittoni ◽

Edoardo Lenci ◽

Alessio Lupi ◽

...

Keyword(s):

Colorectal Cancer ◽

Microsatellite Instability ◽

Mismatch Repair ◽

Cancer Progression ◽

Checkpoint Inhibitors ◽

Tnm Classification ◽

Standard Of Care ◽

First Line Therapy ◽

Combination Strategies ◽

The Impact

Opinion statementAdvanced colorectal cancer (CRC) is a heterogeneous disease, characterized by several subtypes with distinctive genetic and epigenetic patterns. During the last years, immune checkpoint inhibitors (ICIs) have revamped the standard of care of several tumors such as non-small cell lung cancer and melanoma, highlighting the role of immune cells in tumor microenvironment (TME) and their impact on cancer progression and treatment efficacy. An “immunoscore,” based on the percentage of two lymphocyte populations both at tumor core and invasive margin, has been shown to improve prediction of treatment outcome when added to UICC-TNM classification. To date, pembrolizumab, an anti-programmed death protein 1 (PD1) inhibitor, has gained approval as first-line therapy for mismatch-repair-deficient (dMMR) and microsatellite instability-high (MSI-H) advanced CRC. On the other hand, no reports of efficacy have been presented in mismatch-repair-proficient (pMMR) and microsatellite instability-low (MSI-L) or microsatellite stable (MSS) CRC. This group includes roughly 95% of all advanced CRC, and standard chemotherapy, in addition to anti-EGFR or anti-angiogenesis drugs, still represents first treatment choice. Hopefully, deeper understanding of CRC immune landscape and of the impact of specific genetic and epigenetic alterations on tumor immunogenicity might lead to the development of new drug combination strategies to overcome ICIs resistance in pMMR CRC, thus paving the way for immunotherapy even in this subgroup.

Download Full-text

Implementation of a Rapid Phenotypic Susceptibility Platform for Gram-Negative Bloodstream Infections With Paired Antimicrobial Stewardship Intervention: Is the Juice Worth the Squeeze?

Clinical Infectious Diseases ◽

10.1093/cid/ciab126 ◽

2021 ◽

Author(s):

Evan D Robinson ◽

Allison M Stilwell ◽

April E Attai ◽

Lindsay E Donohue ◽

Megan D Shah ◽

...

Keyword(s):

Median Time ◽

Antimicrobial Stewardship ◽

A Priori ◽

Bloodstream Infections ◽

Standard Of Care ◽

Post Intervention ◽

Gram Negative ◽

Stewardship Program ◽

Days Of Therapy ◽

The Impact

Abstract Background Implementation of the Accelerate PhenoTM Gram-negative platform (RDT) paired with antimicrobial stewardship program (ASP) intervention projects to improve time to institutional-preferred antimicrobial therapy (IPT) for Gram-negative bacilli (GNB) bloodstream infections (BSIs). However, few data describe the impact of discrepant RDT results from standard of care (SOC) methods on antimicrobial prescribing. Methods A single-center, pre-/post-intervention study of consecutive, nonduplicate blood cultures for adult inpatients with GNB BSI following combined RDT + ASP intervention was performed. The primary outcome was time to IPT. An a priori definition of IPT was utilized to limit bias and to allow for an assessment of the impact of discrepant RDT results with the SOC reference standard. Results Five hundred fourteen patients (PRE 264; POST 250) were included. Median time to antimicrobial susceptibility testing (AST) results decreased 29.4 hours (P < .001) post-intervention, and median time to IPT was reduced by 21.2 hours (P < .001). Utilization (days of therapy [DOTs]/1000 days present) of broad-spectrum agents decreased (PRE 655.2 vs POST 585.8; P = .043) and narrow-spectrum beta-lactams increased (69.1 vs 141.7; P < .001). Discrepant results occurred in 69/250 (28%) post-intervention episodes, resulting in incorrect ASP recommendations in 10/69 (14%). No differences in clinical outcomes were observed. Conclusions While implementation of a phenotypic RDT + ASP can improve time to IPT, close coordination with Clinical Microbiology and continued ASP follow up are needed to optimize therapy. Although uncommon, the potential for erroneous ASP recommendations to de-escalate to inactive therapy following RDT results warrants further investigation.

Download Full-text