Patient-Derived Glioma Models: From Patients to Dish to Animals

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults associated with a poor survival. Current standard of care consists of surgical resection followed by radiation and chemotherapy. GBMs are highly heterogeneous, having a complex interaction among different cells within the tumor as well as the tumor microenvironment. One of the main challenges in the neuro-oncology field in general, and GBM in particular, is to find an optimum culture condition that maintains the molecular genotype and phenotype as well as heterogeneity of the original tumor in vitro and in vivo. Established cell lines were shown to be a poor model of the disease, failing to recapitulate the phenotype and harboring non-parental genotypic mutations. Given the growing understanding of GBM biology, the discovery of glioma cancer stem-like cells (GSCs), and their role in tumor formation and therapeutic resistance, scientists are turning more towards patient-derived cells and xenografts as a more representative model. In this review, we will discuss the current state of patient-derived GSCs and their xenografts; and provide an overview of different established models to study GBM biology and to identify novel therapeutics in the pre-clinical phase.

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Novel Neonatal Umbilical Catheter Protection and Stabilization Device in In vitro Model of Catheterized Human Umbilical Cords: Effect of Material and Venting on Bacterial Colonization

American Journal of Perinatology ◽

10.1055/s-0039-1700542 ◽

2019 ◽

Author(s):

Lauren S. Y. Wood ◽

Janene H. Fuerch ◽

Carl L. Dambkowski ◽

Eric F. Chehab ◽

Shivani Torres ◽

...

Keyword(s):

Bacterial Colonization ◽

Bacterial Load ◽

Standard Of Care ◽

Adhesive Tape ◽

Human Umbilical Cord ◽

Current Standard ◽

Central Lines ◽

Umbilical Cords

Abstract Objective Umbilical central lines deliver life-saving medications and nutrition for neonates; however, complications associated with umbilical catheters (UCs) occur more frequently than in adults with central lines (i.e., line migration, systemic infection). We have developed a device for neonatal UC protection and stabilization to reduce catheter exposure to bacteria compared with the standard of care: “goal post” tape configuration. This study analyzes the effect of device venting and material on bacterial load of human umbilical cords in vitro. Study Design Catheters were inserted into human umbilical cord segments in vitro, secured with plastic or silicone vented prototype versus tape, and levels of bacterial colonization were compared between groups after 7 days of incubation. Results Nonvented plastic prototype showed increased bacterial load compared with goal post (p = 0.04). Colonization was comparable between the goal post and all vented plastic prototypes (p ≥ 0.30) and when compared with the vented silicone device (p = 1). Conclusion A novel silicone device does not increase external bacterial colonization compared with the current standard of care for line securement, and may provide a safe, convenient alternative to standard adhesive tape for UC stabilization. Future studies are anticipated to establish safety in vivo, alongside benefits such as migration and infection reduction.

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An innovative cyanoacrylate device developed to improve the current standard of care for intravascular catheter securement

The Journal of Vascular Access ◽

10.1177/1129729819872881 ◽

2019 ◽

Vol 21 (3) ◽

pp. 293-299

Author(s):

Amanda Guido ◽

Sheng Zhang ◽

Cheng Yang ◽

Laura Pook

Keyword(s):

Cost Effective ◽

Standard Of Care ◽

Intravascular Catheter ◽

Current Standard ◽

Care Protocol ◽

Intravascular Catheters ◽

Intravascular Devices ◽

Strength And Durability

Introduction: Over one billion intravascular devices are used worldwide, annually. Due to the associated complications with these devices, the development of a reliable yet cost-effective securement technique is extremely important. The purpose of this study is to demonstrate the strength of a novel catheter securement cyanoacrylate for securing peripheral venous catheters, central venous catheters, peripherally inserted central catheters, and all other intravascular catheter types. Materials and methods: An unprecedented in vitro method was performed to quantify and compare the strength of a novel cyanoacrylate product when securing intravascular catheters inserted into prepared porcine skin. In vivo, canine subjects were used to implant various types of catheters. These catheters were secured with a novel catheter securement cyanoacrylate to test the strength and durability while undergoing simulated clinical stresses. Results: In vitro, the catheter securement cyanoacrylate demonstrated superior strength over conventional catheter securement methods as well as other known cyanoacrylates. The catheter securement cyanoacrylate demonstrated the ability to maintain superior strength for up to 7 days. In vivo, the catheter securement cyanoacrylate demonstrated the ability to withstand five weight tugs per hour for a 3-h duration, alone, while securing three types of catheters in canine subjects. Conclusion: This is one of the first studies to provide quantitative data to support the use of cyanoacrylate for intravascular catheter securement. The results from this research suggest that the novel catheter securement cyanoacrylate can be a simple and cost-effective catheter securement device that can improve the current health care protocol for intravascular catheterization.

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Glioblastoma’s Next Top Model: Novel Culture Systems for Brain Cancer Radiotherapy Research

Cancers ◽

10.3390/cancers11010044 ◽

2019 ◽

Vol 11 (1) ◽

pp. 44 ◽

Cited By ~ 18

Author(s):

Seamus Caragher ◽

Anthony J. Chalmers ◽

Natividad Gomez-Roman

Keyword(s):

Mouse Models ◽

Standard Of Care ◽

Primary Brain Tumor ◽

Current Standard ◽

Meaningful Improvement ◽

Nutrient Sources ◽

Culture Systems ◽

Novel Treatments ◽

Wide Range

Glioblastoma (GBM), the most common and aggressive primary brain tumor in adults, remains one of the least treatable cancers. Current standard of care—combining surgical resection, radiation, and alkylating chemotherapy—results in a median survival of only 15 months. Despite decades of investment and research into the development of new therapies, most candidate anti-glioma compounds fail to translate into effective treatments in clinical trials. One key issue underlying this failure of therapies that work in pre-clinical models to generate meaningful improvement in human patients is the profound mismatch between drug discovery systems—cell cultures and mouse models—and the actual tumors they are supposed to imitate. Indeed, current strategies that evaluate the effects of novel treatments on GBM cells in vitro fail to account for a wide range of factors known to influence tumor growth. These include secreted factors, the brain’s unique extracellular matrix, circulatory structures, the presence of non-tumor brain cells, and nutrient sources available for tumor metabolism. While mouse models provide a more realistic testing ground for potential therapies, they still fail to account for the full complexity of tumor-microenvironment interactions, as well as the role of the immune system. Based on the limitations of current models, researchers have begun to develop and implement novel culture systems that better recapitulate the complex reality of brain tumors growing in situ. A rise in the use of patient derived cells, creative combinations of added growth factors and supplements, may provide a more effective proving ground for the development of novel therapies. This review will summarize and analyze these exciting developments in 3D culturing systems. Special attention will be paid to how they enhance the design and identification of compounds that increase the efficacy of radiotherapy, a bedrock of GBM treatment.

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39. CHARACTERIZING NOVEL INHIBITORS OF BRAIN METASTASIS-INITIATING CELLS

Neuro-Oncology Advances ◽

10.1093/noajnl/vdaa073.027 ◽

2020 ◽

Vol 2 (Supplement_2) ◽

pp. ii7-ii7

Author(s):

Agata Kieliszek ◽

Chitra Venugopal ◽

Blessing Bassey-Archibong ◽

Nikoo Aghaei ◽

Fred Lam ◽

...

Keyword(s):

At Risk ◽

Brain Metastasis ◽

Primary Tumor ◽

Unmet Need ◽

Standard Of Care ◽

Current Standard ◽

One Year ◽

Pdx Models

Abstract BACKGROUND The incidence of brain metastases (BM) is tenfold higher than primary brain tumors. BM commonly originate from primary lung, breast, and melanoma tumors with a 90% mortality rate within one year of diagnosis. Current standard of care for BM includes surgical resection with concurrent chemoradiation, but does not extend median survival past 16 months, posing a large unmet need to identify novel therapies against BM. METHODS From a large in-house biobank of patient-derived BM cell lines, the Singh Lab has generated murine orthotopic patient-derived xenograft (PDX) models of lung, breast, and melanoma BM that recapitulate the stages of BM progression as seen in human patients. Using these three PDX models, we identified a population of “pre-metastatic” brain metastasis-initiating cells (BMICs) that are newly arrived in the brain but have yet to form detectable tumors. Pre-metastatic BMICs are not detectable in human patients but are important therapeutic targets with the potential to prevent BM in at-risk patients. RESULTS RNA sequencing of pre-metastatic BMICs from all three PDX primary tumor models with subsequent Connectivity Map analysis identified novel compounds that have the potential of killing all three types of BMICs. In particular, we identified two compounds that have selective killing of BMICs in vitro from all three primary tumor cohorts while sparing non-cancerous cells. We further characterized their ability to inhibit the self-renewal and proliferative properties of BMICs. Ongoing in vivo work will investigate the compounds’ preclinical utilities in preventing BM. CONCLUSION Identification of novel small molecules that target BMICs could prevent the formation of BM completely and dramatically improve the prognosis of at-risk cancer patients.

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Temozolomide antagonizes oncolytic immunovirotherapy in glioblastoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2019-000345 ◽

2020 ◽

Vol 8 (1) ◽

pp. e000345 ◽

Cited By ~ 3

Author(s):

Dipongkor Saha ◽

Samuel D Rabkin ◽

Robert L Martuza

Keyword(s):

Dna Methyltransferase ◽

Standard Of Care ◽

Interleukin 12 ◽

Current Standard ◽

Antitumor Effects ◽

Methylguanine Dna Methyltransferase ◽

Beneficial Effects ◽

Simplex Virus

BackgroundTemozolomide (TMZ) chemotherapy is a current standard of care for glioblastoma (GBM), however it has only extended overall survival by a few months. Because it also modulates the immune system, both beneficially and negatively, understanding how TMZ interacts with immunotherapeutics is important. Oncolytic herpes simplex virus (oHSV) is a new class of cancer therapeutic with both cytotoxic and immunostimulatory activities. Here, we examine the combination of TMZ and an oHSV encoding murine interleukin 12, G47Δ-mIL12, in a mouse immunocompetent GBM model generated from non-immunogenic 005 GBM stem-like cells (GSCs.MethodsWe first investigated the cytotoxic effects of TMZ and/or G47Δ-IL12 treatments in vitro, and then the antitumor effects of combination therapy in vivo in orthotopically implanted 005 GSC-derived brain tumors. To improve TMZ sensitivity, O6-methylguanine DNA methyltransferase (MGMT) was inhibited. The effects of TMZ on immune cells were evaluated by flow cytometery and immunohistochemistry.ResultsThe combination of TMZ+G47Δ-IL12 kills 005 GSCs in vitro better than single treatments. However, TMZ does not improve the survival of orthotopic tumor-bearing mice treated with G47Δ-IL12, but rather can abrogate the beneficial effects of G47Δ-IL12 when the two are given concurrently. TMZ negatively affects intratumor T cells and macrophages and splenocytes. Addition of MGMT inhibitor O6-benzylguanine (O6-BG), an inactivating pseudosubstrate of MGMT, to TMZ improved survival, but the combination with G47Δ-IL12 did not overcome the antagonistic effects of TMZ treatment on oHSV therapy.ConclusionsThese results illustrate that chemotherapy can adversely affect oHSV immunovirotherapy. As TMZ is the standard of care for GBM, the timing of these combined therapies should be taken into consideration when planning oHSV clinical trials with chemotherapy for GBM.

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CBMT-42. A GENOME-WIDE CRISPR-Cas9 SCREEN FOR GENES REGULATING QUIESCENT-LIKE STATES IN GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noz175.164 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi42-vi42

Author(s):

Heather Feldman ◽

Anca Mihalas ◽

Christopher Plaisier ◽

Anoop Patel ◽

Patrick Paddison

Keyword(s):

Tumor Cells ◽

Standard Of Care ◽

Cell Types ◽

Phenotypic Characterization ◽

Current Standard ◽

Genome Wide ◽

A Genome ◽

G0 Phase

Abstract Current standard of care therapy for glioblastoma (GBM) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, non-cycling, quiescent-like states (G0 phase cells) are present in both normal tissue and tumors and play important roles in maintaining heterogeneity and cellular hierarchies. The presence of quiescent-like/G0 states therefore represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. However, it remains largely unclear to what degree or by what mechanisms tumor cells enter into or exit from quiescent-like states. To gain insight into how glioblastoma cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) harboring a p27-mVenus reporter construct, which is stabilized when cells enter a G0-like state. By assaying p27 reporteractivity, we were able to identify sgRNAs enriched in p27hipopulations and, which upon retest, trigger a G0-like arrest in GSCs. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, including KAT5 itself. Remarkably, we show that downregulation of KAT5 in vitro and in vivo dramatically increases the pool of cells in G0-like states in GSC cultures and GSC-induced tumors. Using single cell RNA-sequencing, we show that this cell state is characterized by gene expression signatures similar to those found in non-dividing subpopulations of GBM tumors and quiescent neural stem cells. In addition, we perform in-depth molecular and phenotypic characterization of these induced G0-like states, including epigenetic and metabolic profiles. These suggest a key role for KAT5 in regulating genes related to protein synthesis. In summary, our results suggest that Tip60/KAT5 activity plays key roles in G0 ingress/egress for GBM tumors and may provide novel therapeutic opportunities.

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Analogs of marinopyrrole A show enhancement to observed in vitro potency against acute Toxoplasma gondii infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00794-21 ◽

2021 ◽

Author(s):

Matthew C. Martens ◽

Yan Liu ◽

Austin G. Sanford ◽

Alexander I. Wallick ◽

Rosalie C. Warner ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Acute Infection ◽

Standard Of Care ◽

Host Cells ◽

Infection Model ◽

Current Standard ◽

Clinical Consequences ◽

Toxoplasma Gondii Infection

The apicomplexan parasite Toxoplasma gondii is the causative agent of toxoplasmosis, a globally distributed infection with severe clinical consequences for immunocompromised individuals and developing fetuses. There are few available treatments, and these are associated with potentially severe adverse effects. Marinopyrrole A, a compound discovered in a marine Streptomyces species, has previously been found to exhibit potent antimicrobial activity, prompting our interest in exploring efficacy against Toxoplasma gondii . We found that marinopyrrole A was a highly potent anti- Toxoplasma molecule, with an in vitro 50% maximal inhibitory concentration (IC 50 ) of 0.31 μM corresponding to a higher potency than that of the current standard of care (pyrimethamine); however, addition of 20% serum led to abrogation of potency, and toxicity to human cell lines was observed. Yet, application of marinopyrrole A to an in vivo lethal acute infection model facilitated significantly enhanced survival at doses of 5, 10, and 20 mg/kg. We then tested a series of marinopyrrole A analogs—RL002, RL003, and RL125—demonstrating significantly increased potency in vitro , with IC 50 values ranging from 0.09-0.17 μM (3.6-6.8X increase relative to pyrimethamine). No detectable cytotoxicity was observed up to 50 μM in human foreskin fibroblasts, with cytotoxicity in HepG2 cells ranging from ∼28-50 μM, corresponding to >200X selectivity for parasites over host cells. All analogs additionally showed reduced sensitivity to serum. Further, RL003 potently inhibited in vitro -generated bradyzoites at 0.245 μM. Taken together, these data support further development of marinopyrrole A analogs as promising anti- Toxoplasma molecules to further combat this prevalent infection.

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VIS832, a novel CD138-targeting monoclonal antibody, potently induces killing of human multiple myeloma and further synergizes with IMiDs or bortezomib in vitro and in vivo

Blood Cancer Journal ◽

10.1038/s41408-020-00378-z ◽

2020 ◽

Vol 10 (11) ◽

Author(s):

Tengteng Yu ◽

Bharat Chaganty ◽

Liang Lin ◽

Lijie Xing ◽

Boopathy Ramakrishnan ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

Standard Of Care ◽

Optimal Dose ◽

Current Standard ◽

Effector Cells ◽

Human Multiple Myeloma ◽

Target Binding

Abstract Therapeutically targeting CD138, a define multiple myeloma (MM) antigen, is not yet approved for patients. We here developed and determined the preclinical efficacy of VIS832, a novel therapeutic monoclonal antibody (MoAb) with differentiated CD138 target binding to BB4 that is anti-CD138 MoAb scaffold for indatuximab ravtansine (BT062). VIS832 demonstrated enhanced CD138-binding avidity and significantly improved potency to kill MM cell lines and autologous patient MM cells regardless of resistance to current standard-of-care therapies, via robust antibody-dependent cellular cytotoxicity and phagocytosis mediated by NK and macrophage effector cells, respectively. Specifically, CD38-targeting daratumumab-resistant MM cells were highly susceptible to VIS832 which, unlike daratumumab, spares NK cells. Superior maximal cytolysis of VIS832 vs. daratumumab corresponded to higher CD138 vs. CD38 levels in MM cells. Furthermore, VIS832 acted synergistically with lenalidomide or bortezomib to deplete MM cells. Importantly, VIS832 at a sub-optimal dose inhibited disseminated MM1S tumors in vivo as monotherapy (P < 0.0001), and rapidly eradicated myeloma burden in all mice concomitantly receiving bortezomib, with 100% host survival. Taken together, these data strongly support clinical development of VIS832, alone and in combination, for the therapeutic treatment of MM in relapsed and refractory patients while pointing to its potential therapeutic use earlier in disease intervention.

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STEM-01. TARGETING BRAIN METASTASIS-INITIATING CELLS: A PREVENTATIVE APPROACH

Neuro-Oncology ◽

10.1093/neuonc/noaa215.818 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii196-ii196

Author(s):

Agata Kieliszek ◽

Chitra Venugopal ◽

Blessing Bassey-Archibong ◽

Fred Lam ◽

Sheila Singh ◽

...

Keyword(s):

At Risk ◽

Brain Metastasis ◽

Primary Tumor ◽

Unmet Need ◽

Standard Of Care ◽

Current Standard ◽

One Year ◽

Pdx Models

Abstract BACKGROUND The incidence of brain metastases (BM) is tenfold higher than primary brain tumors. BM commonly originate from primary lung, breast, and melanoma tumors with a 90% mortality rate within one year of diagnosis. Current standard of care for BM includes surgical resection with concurrent chemoradiation, but does not extend median survival past 16 months, posing a large unmet need to identify novel therapies against BM. METHODS From a large in-house biobank of patient-derived BM cell lines, the Singh Lab has generated murine orthotopic patient-derived xenograft (PDX) models of lung, breast, and melanoma BM that recapitulate the stages of BM progression as seen in humans. Using these three PDX models, we identified a population of “pre-metastatic” brain metastasis-initiating cells (BMICs) that are newly arrived in the brain but have yet to form detectable tumors. Pre-metastatic BMICs are not detectable in human patients but are important therapeutic targets with the potential to prevent BM in at-risk patients. RESULTS RNA sequencing of pre-metastatic BMICs from all three PDX primary tumor models with subsequent Connectivity Map analysis identified novel compounds that have the potential of killing all three types of BMICs. In particular, we identified two compounds that have selective killing of BMICs in vitro from all three primary tumor cohorts while sparing non-cancerous cells. We further characterized their ability to inhibit the self-renewal and proliferative properties of BMICs. Ongoing in vivo work will investigate the compounds’ preclinical utilities in preventing BM. CONCLUSION Identification of novel small molecules that target BMICs could prevent the formation of BM completely and dramatically improve the prognosis of at-risk cancer patients.

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EXTH-46. ALISERTIB POTENTIATES THE EFFECT OF CABOZANTINIB AND VANDETANIB AGAINST GLIOBLASTOMA CELLS

Neuro-Oncology ◽

10.1093/neuonc/noz175.378 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi92-vi92

Author(s):

Brian Williams ◽

Cory Zumbar ◽

Caroline Mifsud ◽

Kendall Smith ◽

Muge Sak ◽

...

Keyword(s):

Clinical Trials ◽

Single Agent ◽

Annexin V ◽

Standard Of Care ◽

In Vivo Studies ◽

Vegf Receptor ◽

Glioblastoma Cell ◽

Current Standard

Abstract Glioblastoma is the most common primary malignant brain tumor in adults and has a poor prognosis with current standard of care. Aurora-A (AURKA) is a serine-threonine kinase critical for mitotic progression through its role in centrosome maturation and mitotic spindle assembly. Vascular endothelial growth factor (VEGF) is a regulator of tumor angiogenesis and vasculogenesis. Both AURKA and VEGF are commonly over-expressed in glioblastoma. The AURKA inhibitor alisertib and inhibitors of VEGF have been shown to inhibit glioblastoma cell proliferation in vitro and in vivo, and both have been used in clinical trials for glioblastoma. Here we tested the ability of alisertib to potentiate the effects of the VEGF receptor (VEGFR) inhibitors cabozantinib and vandetanib using colony formation assays in U1242 and U87 glioblastoma cell lines. Chou-Talalay and Bliss independence models demonstrated synergistic growth inhibition in some cases. Annexin V binding assays were performed to assess the extent that apoptosis could account for the inhibitory effects in U87 cells, and a concentration of alisertib that did not cause apoptosis as a single agent was found to increase the induction of apoptosis caused by cabozantinib. These results support the rationale for further in vitro and in vivo studies of the combined use of alisertib and VEGFR inhibitors, including possible future clinical trials.

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