TMOD-31. AN ORGANOTYPIC TISSUE PLATFORM TO BRIDGE IN VITRO AND IN VIVO ASSAYS FOR BRAIN CANCER TREATMENT

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi222-vi222
Author(s):  
Breanna Mann ◽  
Noah Bell ◽  
Denise Dunn ◽  
Scott Floyd ◽  
Shawn Hingtgen ◽  
...  
Keyword(s):  
Cell Lines ◽  
Brain Cancer ◽  
Brain Slice ◽  
Brain Slices ◽  
In Vitro Assays ◽  
Preclinical Model ◽  
Short Period ◽  
The Brain

Abstract Brain cancers remain one of the greatest medical challenges. The lack of experimentally tractable models that recapitulate brain structure/function represents a major impediment. Platforms that enable functional testing in high-fidelity models are urgently needed to accelerate the identification and translation of therapies to improve outcomes for patients suffering from brain cancer. In vitro assays are often too simple and artificial while in vivo studies can be time-intensive and complicated. Our live, organotypic brain slice platform can be used to seed and grow brain cancer cell lines, allowing us to bridge the existing gap in models. These tumors can rapidly establish within the brain slice microenvironment, and morphologic features of the tumor can be seen within a short period of time. The growth, migration, and treatment dynamics of tumors seen on the slices recapitulate what is observed in vivo yet is missed by in vitro models. Additionally, the brain slice platform allows for the dual seeding of different cell lines to simulate characteristics of heterogeneous tumors. Furthermore, live brain slices with embedded tumor can be generated from tumor-bearing mice. This method allows us to quantify tumor burden more effectively and allows for treatment and retreatment of the slices to understand treatment response and resistance that may occur in vivo. This brain slice platform lays the groundwork for a new clinically relevant preclinical model which provides physiologically relevant answers in a short amount of time leading to an acceleration of therapeutic translation.

scholarly journals BSCI-12. Inhibition of melanoma brain metastasis by targeting miR-146a

2021 ◽  
Vol 3 (Supplement_3) ◽  
pp. iii3-iii3
Author(s):  
Jiwei Wang ◽  
Emma Rigg ◽  
Taral R Lunavat ◽  
Wenjing Zhou ◽  
Zichao Feng ◽  
...  
Keyword(s):  
Brain Metastasis ◽  
Tumor Cells ◽  
Cell Lines ◽  
Western Blots ◽  
Cell Growth And Migration ◽  
Human Astrocytes ◽  
And Migration ◽  
The Brain

Abstract Background Melanoma has the highest propensity of any cancer to metastasize to the brain, with late-stage patients developing brain metastasis (MBM) in 40% of cases. Survival of patients with MBM is around 8 months with current therapies, illustrating the need for new treatments. MBM development is likely caused by molecular interactions between tumor cells and the brain, constituting the brain metastatic niche. miRNAs delivered by exosomes released by the primary tumor cells may play a role in niche establishment, yet the mechanisms are poorly understood. Here, the aim was to identify miRNAs released by exosomes from melanomas, which may be important in niche establishment and MBM progression. Materials and Methods miRNAs from exosomes collected from human astrocytes, melanocytes, and MBM cell lines were profiled to determine differential expression. Functional in vitro validation was performed by cell growth and migration assays, cytokine arrays, qPCR and Western blots. Functional in vivo studies were performed after miR knockdown in MBM cell lines. An in silico docking study was performed to determine drugs that potentially inhibit transcription of miR-146a to impede MBM development. Results miR-146a was the most upregulated miRNA in exosomes from MBM cells and was highly expressed in human and animal MBM samples. miR-146a mimics activated human astrocytes, shown by increased proliferation and migration, elevated expression of GFAP in vitro and in mouse brain tumor samples, and increased cytokine production. In animal studies, knockdown of miR-146a in MBM cells injected intracardially into mice reduced BM burden and increased animal survival. Based on the docking studies, deserpidine was found to be an effective inhibitor of MBM growth in vitro and in vivo. Conclusions MiR-146a may play an important role in MBM development, and deserpidine is a promising candidate for clinical use.

P16.08 Inhibition of melanoma brain metastasis by targeting miR-146a

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii57-ii57
Author(s):  
J Wang ◽  
E K Rigg ◽  
T R Lunavat ◽  
W Zhou ◽  
Z Feng ◽  
...  
Keyword(s):  
Brain Metastasis ◽  
Tumor Cells ◽  
Cell Lines ◽  
Western Blots ◽  
Cell Growth And Migration ◽  
Human Astrocytes ◽  
And Migration ◽  
The Brain

Abstract BACKGROUND Melanoma has the highest propensity of any cancer to metastasize to the brain, with late-stage patients developing brain metastasis (MBM) in 40% of cases. Survival of patients with MBM is around 8 months with current therapies, illustrating the need for new treatments. MBM development is likely caused by molecular interactions between tumor cells and the brain, constituting the brain metastatic niche. miRNAs delivered by exosomes released from the primary tumor cells may play a role in niche establishment, yet the mechanisms are poorly understood. Here, the aim was to identify miRNAs released by exosomes from melanomas, which may be important in niche establishment and MBM progression. MATERIAL AND METHODS miRNAs in exosomes collected from human astrocytes, melanocytes, and MBM cell lines were profiled to determine differential expression. Functional in vitro validation was performed by cell growth and migration assays, cytokine arrays, qPCR and Western blots. Functional in vivo studies were performed after miR knockdown in MBM cell lines. An in silico docking study was performed to determine drugs that potentially inhibit transcription of miR-146a to impede MBM development. RESULTS miR-146a was the most upregulated miRNA in exosomes from MBM cells and was highly expressed in human and animal MBM samples. miR-146a mimics activated human astrocytes, shown by increased proliferation and migration, elevated expression of GFAP in vitro and in mouse brain tumor samples, and increased cytokine production. In animal studies, knockdown of miR-146 in MBM cells injected intracardially into mice reduced BM burden and increased animal survival. Based on the docking studies, deserpidine was found to be an effective inhibitor of MBM growth in vitro and in vivo. CONCLUSION miR-146a may play an important role in MBM development, and deserpidine is a promising candidate for clinical use.

scholarly journals Targeted Brain Tumor Therapy by Inhibiting the MDM2 Oncogene: In Vitro and In Vivo Antitumor Activity and Mechanism of Action

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1592
Author(s):  
Surendra R. Punganuru ◽  
Viswanath Arutla ◽  
Wei Zhao ◽  
Mehrdad Rajaei ◽  
Hemantkumar Deokar ◽  
...  
Keyword(s):  
Brain Tumor ◽  
Antitumor Activity ◽  
Cell Lines ◽  
Tumor Therapy ◽  
Single Agent ◽  
M Cell ◽  
In Vivo Antitumor Activity ◽  
The Brain

There is a desperate need for novel and efficacious chemotherapeutic strategies for human brain cancers. There are abundant molecular alterations along the p53 and MDM2 pathways in human glioma, which play critical roles in drug resistance. The present study was designed to evaluate the in vitro and in vivo antitumor activity of a novel brain-penetrating small molecule MDM2 degrader, termed SP-141. In a panel of nine human glioblastoma and medulloblastoma cell lines, SP-141, as a single agent, potently killed the brain tumor-derived cell lines with IC50 values ranging from 35.8 to 688.8 nM. Treatment with SP-141 resulted in diminished MDM2 and increased p53 and p21cip1 levels, G2/M cell cycle arrest, and marked apoptosis. In intracranial xenograft models of U87MG glioblastoma (wt p53) and DAOY medulloblastoma (mutant p53) expressing luciferase, treatment with SP-141 caused a significant 4- to 9-fold decrease in tumor growth in the absence of discernible toxicity. Further, combination treatment with a low dose of SP-141 (IC20) and temozolomide, a standard anti-glioma drug, led to synergistic cell killing (1.3- to 31-fold) in glioma cell lines, suggesting a novel means for overcoming temozolomide resistance. Considering that SP-141 can be taken up by the brain without the need for any special delivery, our results suggest that SP-141 should be further explored for the treatment of tumors of the central nervous system, regardless of the p53 status of the tumor.

Acute hypoxia induces elevation of ornithine decarboxylase activity in neonatal rat brain slices

1995 ◽  
Vol 7 (3) ◽  
pp. 385 ◽  
Author(s):  
LD Longo ◽  
S Packianathan
Keyword(s):  
Rat Brain ◽  
Ornithine Decarboxylase ◽  
Bovine Serum ◽  
Brain Slice ◽  
Brain Slices ◽  
Neonatal Rat ◽  
Newborn Rat ◽  
Rat Brain Slice

Recent studies in vivo have demonstrated that ornithine decarboxylase (ODC) activity in the fetal rat brain is elevated 4-5-fold by acute maternal hypoxia. This hypoxic-associated increase is seen in the rat brain in both the newborn and the adult. Because of the intimate involvement of ODC in transcription and translation, as well as in growth and development, it is imperative that the manner in which hypoxia affects the regulation of this enzyme be better understood. In order to achieve this, a brain preparation in vitro was required to eliminate the confounding effects of the dam on the fetal and newborn brain ODC activity in vivo. Therefore, brain slices from 3-4-day-old (P-3) newborn rats were utilized to test the hypothesis that ODC activity increases in response to hypoxia in vitro. Cerebral slices from the P-3 rat pups were allowed to equilibrate and recover in artificial cerebrospinal fluid (ACSF) continuously bubbled with a mixture of 95% O2 and 5% CO2 for 1 h before beginning hypoxic exposures. Higher basal ODC activities were obtained by treating the slices with 0.03% fetal bovine serum (FBS) and 0.003% bovine serum albumin (BSA), rather than with ACSF alone. Hypoxia was induced in the slices by replacing the gas with 40%, 21%, 10%, or 5% O2, all with 5% CO2 and balance N2. With FBS and BSA treatment, ODC activity was maintained at about 0.15-0.11 nM CO2 mg-1 protein h-1 throughout the experiment, which was 2-3-fold higher than that without FBS and BSA. ODC activity increased significantly and peaked between 1 h and 2 h after initiation of hypoxia. For instance, with 21% O2, ODC activity increased approximately 1.5-fold at 1 h and approximately 2-fold at 2 h. These studies demonstrate that: (1) the hypoxic-induced increases observed in vivo in the fetal and newborn rat brain ODC activity can be approximated in a newborn rat brain slice preparation in vitro; (2) newborn rat brain slice preparations may provide an alternative to methods in vivo or cell culture methods for studying the regulation of acute hypoxic-induced enzymes; and (3) high, stable baseline ODC activities in brain slices suggest that the cells in the slice are capable of active metabolism if FBS and BSA are available to mimic conditions in vivo.

A novel orthotopic animal model of human chondrosarcoma

2012 ◽  
Vol 21 (04) ◽  
pp. 296-300
Author(s):  
K. Horas ◽  
M. Tonak ◽  
A. A. Kurth
Keyword(s):  
Animal Model ◽  
Cell Lines ◽  
Bone Tumour ◽  
In Vivo Studies ◽  
Preclinical Model ◽  
Orthotopic Implantation ◽  
Chondrosarcoma Cell ◽  
Human Chondrosarcoma

SummaryChondrosarcoma is the second most common primary malignant bone tumour in humans. Currently, surgical resection is the only appropriate curative approach as it is relatively unresponsive to traditional chemoand radiotherapy. However, a complete resection is often hindered due to the proximity to organs resulting in a poor outcome of this challenging malignancy. Few novel antitumour agents have been tested on different chondrosarcoma cell lines in vitro so far. In order to qualify new agents in vivo, animal models are often used in which cell lines are subcutaneously injected prior to chemotherapeutical treatment. These types of models often lack relevance to the human chondrosarcoma as the number of agents that fail in the clinic far outweighs those considered effective on in vivo studies. Orthotopic xenograft models however are of much more predictive value. Thus, the development of a novel orthotopic animal model for human chondrosarcoma using a three-dimensional matrix carrying tumour cells, was the aim of this study. For that purpose, SW-1353, a human bone chondrosarcoma cell line, was first cultured in MatrigelTM, followed by orthotopic implantation into10 SCID mice by intra-tibial injection. After 40 days, the animals developed localized bone tumours verified by radiographic and histological examinations. Radiologic and histological sections showed osteolysis and invasive tumour growth. This study demonstrates a promising new method for effective and reproducible orthotopic implantation of human chondrosarcoma. The presented animal model allows further examination and can be used as a predictive preclinical model for anticancer drug activity in humans.

Biochemistry

1936 ◽  
Vol 82 (339) ◽  
pp. 431-433
Author(s):  
J. H. Quastel
Keyword(s):  
Nervous System ◽  
Brain Tissue ◽  
Brain Slice ◽  
Brain Slices ◽  
Ringer Solution ◽  
Living Animal ◽  
Dominant Form ◽  
Straight Line ◽  
The Brain

I want to speak of the work we have been doing in Cardiff on the metabolism of the nervous system. The work was carried out there because of the importance of the narcosis treatment. It seemed to us there a pity that a treatment such as that should be given up because of the considerable toxicity possible in relation to it. The research was undertaken to see if we could diminish the toxicity, at the same time seeking an idea as to how narcotics work. I ask that you will realize that the main substance burned by the brain is glucose. The dominant form of metabolism in the nervous system is connected with the breakdown of glucose and lactic acid, and this can be proved by experiment in the living animal and with brain-tissue in vitro. In doing experiments we are not able to carry out work with human brain, because we cannot get human tissue fresh enough, so we have to carry out experiments with animals. They are carried out in this way. We cut slices of the cortex of the brain as soon as the animal is dead, that is to say, within ten minutes of death the brain is out and slices have been cut. They are placed in a physiological medium in the presence of glucose, and we follow the metabolism of that tissue, which allows us to estimate the amount of oxygen being taken up by the brain. If luminal, chloretone, hyoscine or somnifaine be placed with the brain-tissue, then the respiration, instead of being at the usual level, starts lower down, and maintains a straight line. We wanted to see whether this action is reversible or irreversible. If the latter, then on removing the brain-slices from the narcotic it should no longer behave like a normal piece of tissue. Actually, when the brain-slice is removed and placed in Ringer solution, with no narcotic, the respiration goes up and becomes equal to that shown by the slice which had no narcotic. That is to say, the process is reversible.

EXTH-78. WP1874, A HIGHLY POTENT UNIQUE DNA BINDING AGENT WITH ENHANCED CNS UPTAKE

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi181-vi181
Author(s):  
Rafal Zielinski ◽  
Roberto Cardenas-Zuniga ◽  
Maria Poimenidou ◽  
Magdalena Remiszewski ◽  
Edd Felix ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Lines ◽  
Water Solubility ◽  
Intraperitoneal Administration ◽  
Orthotopic Model ◽  
Biodistribution Studies ◽  
Drug Discovery Program ◽  
The Brain

Abstract As part of our drug discovery program, we have developed structure-based modular designs of unique DNA-binding agents. The approach combines DNA intercalating and DNA “minor-groove-binding” modules. We have discovered compound WP1244 that potentially binds up to 10 bp long sequences of DNA. The unique and intriguing feature of WP1244 is its high CNS uptake combined with the picomolar to low nanomolar cytotoxicity against ependymoma and glioblastoma multiforme (GBM) cell lines and demonstrated in vivo activity in the orthotopic model of GBM. To improve water solubility and develop an IV formulation, we have synthesized WP1874, a mesylate salt of WP1244, and initiated its preclinical characterization. WP1874, similarly to its parental compound, shows high cytotoxicity in ependymoma, GBM, and medulloblastoma cell lines with IC50 in low nanomolar range and it was up to 100 to 200 times more potent than doxorubicin. Interestingly, WP1874 does not appear to be cytotoxic against normal kidney cells (VeroC1008) with IC50 > 10 μM. Preliminary pharmacokinetic and biodistribution studies performed in CD-1 mice with intact brains revealed enhanced penetration of WP1874 to the brain with Cmax 1.5-fold greater than in plasma. Respectively, WP1874 Cmax in the brain was 2.3 ug/g (~2.0 μM) vs. 1.5 μg/ml (1.3 μM) in plasma. Acute toxicity in intravenously administered WP1874 was LD50 >15mg/kg. No mortalities or any apparent toxicity symptoms were recorded for six intravenous weekly doses of WP1874 at 2.5 or 5 mg/kg in CD-1, Balb/c, or nude athymic mice. Intraperitoneal administration was well-tolerated up to 5 mg/kg given three times a week for four cycles. High CNS uptake, excellent cytotoxicity against different brain cancer cell lines, and low toxicity in vivo and in vitro against normal cells warrant further investigation of WP1874 as a mechanically unique potential anticancer agent against CNS malignancies.

The HIV-Protease Inhibitor Ritonavir(Norvir®) Is Active Against Human AML Blasts In Vitro and In Vivo and Has Synergistic Cytotoxic Activity with the Proteaseome Inhibitor Bortezomib (Velcade®).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2464-2464
Author(s):  
Christoph Driessen ◽  
Müller-Ide Hendrik ◽  
Gogel Jeanette ◽  
Kraus Marianne ◽  
Kanz Lothar ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Cell Lines ◽  
Single Agent ◽  
Proteasome Inhibition ◽  
Hiv Protease ◽  
Malignant Cells ◽  
Short Period ◽  
Proteolytic Pathways

Abstract Upregulation of alternative proteolytic pathways characterizes malignant cells that overcome proteasome inhibition in vitro. The proteasome inhibitor Bortezomib (Velcade®), which selectively targets only one of the three active subunits of the proteasome, has shown limited activity in AML. Ritonavir (Norvir®) is an aspartate protease inhibitor used in intensive HIV-therapy, where therapeutic levels of 5–20μM are reached with an oral dose of 1200 mg/d. A cytotoxic effect of Ritonavir against malignant cells due to proteasome inhibition has been suggested (Gaedicke et al., Cancer Research 62, December 1, 2002). We have here tested the effect of Ritonavir on AML cells, both as single agent and in combination with Bortezomib. Ritonavir induced cytotoxic death in AML cell lines and primary AML blasts with an IC50 of 30–40 μM in vitro. The combination of Ritonavir and Bortezomib was synergistic in vitro, i.e. subtoxic concentrations of Ritonavir at 10 μM combined with subtoxic Bortezomib 5–10 nM induced robust cytotoxicity in AML cell lines and freshly isolated primary AML blasts. Using a novel chemical probe that for the first time allows to visualize the individual activity of proteasomal subunits in intact AML blasts, we show that Velcade selectively abrogates β5 proteasomal activity at 20 nM in AML cells, as expected. Ritonavir, by contrast, had no effect on active proteasomal subunits up to 50 μM. Thus, the synergistic effect of Ritonavir with Bortezomib on AML cells is not due to inhibition of the same proteasomal target by both drugs, but more likely mediated by blocking alternative proteolytic pathways. One individual patient aged 72 years with an early relaps of AML was treated with Ritonavir 400–600 mg/d p.o.. During treatment, the absolute leukocyte count dropped from 24000/μl to 8000/μl while the ANC raised from 185/μl to 1530/μl. Ritonavir was withdrawn due to diarrhoea and abdominal cramps, leading to a sharp increase in peripheral blood blasts and leukocytes. Retreatment with Ritonavir at a reduced dose of 200 mg/d combined with Velcade 1mg/sqm was tolerated and stabilized leukocyte counts for a short period of time. We conclude that Ritonavir has activity against chemotherapy-refractory AML in vitro and in vivo. The combination of Velcade and Ritonavir might allow to synergistically target the proteolytic machinery of AML blasts with tolerable toxicity.

scholarly journals Noggin rescues age-related stem cell loss in the brain of senescent mice with neurodegenerative pathology

2018 ◽  
Vol 115 (45) ◽  
pp. 11625-11630 ◽  
Author(s):  
María Díaz-Moreno ◽  
Tomás Armenteros ◽  
Simona Gradari ◽  
Rafael Hortigüela ◽  
Laura García-Corzo ◽  
...  
Keyword(s):  
Healthy Aging ◽  
Late Onset ◽  
Hippocampal Neurogenesis ◽  
Bmp Signaling ◽  
In Vitro Assays ◽  
Age Related ◽  
Samp8 Mice ◽  
The Brain

Increasing age is the greatest known risk factor for the sporadic late-onset forms of neurodegenerative disorders such as Alzheimer’s disease (AD). One of the brain regions most severely affected in AD is the hippocampus, a privileged structure that contains adult neural stem cells (NSCs) with neurogenic capacity. Hippocampal neurogenesis decreases during aging and the decrease is exacerbated in AD, but the mechanistic causes underlying this progressive decline remain largely unexplored. We here investigated the effect of age on NSCs and neurogenesis by analyzing the senescence accelerated mouse prone 8 (SAMP8) strain, a nontransgenic short-lived strain that spontaneously develops a pathological profile similar to that of AD and that has been employed as a model system to study the transition from healthy aging to neurodegeneration. We show that SAMP8 mice display an accelerated loss of the NSC pool that coincides with an aberrant rise in BMP6 protein, enhanced canonical BMP signaling, and increased astroglial differentiation. In vitro assays demonstrate that BMP6 severely impairs NSC expansion and promotes NSC differentiation into postmitotic astrocytes. Blocking the dysregulation of the BMP pathway and its progliogenic effect in vivo by intracranial delivery of the antagonist Noggin restores hippocampal NSC numbers, neurogenesis, and behavior in SAMP8 mice. Thus, manipulating the local microenvironment of the NSC pool counteracts hippocampal dysfunction in pathological aging. Our results shed light on interventions that may allow taking advantage of the brain’s natural plastic capacity to enhance cognitive function in late adulthood and in chronic neurodegenerative diseases such as AD.

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