scholarly journals DDRE-02. THERAPEUTIC TARGETING OF BRAIN METASTASIS WITH ERK INHIBITOR LY3214996 USING A NOVEL IN VIVO MODEL OF LUNG-TO-BRAIN METASTASIS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii61-ii61
Author(s):  
Mohini Singh ◽  
Naema Nayyar ◽  
Priscilla Brastianos
Keyword(s):  
Brain Metastasis ◽  
Brain Metastases ◽  
Lung Tumor ◽  
In Vivo Model ◽  
Post Inoculation ◽  
Durable Response ◽  
Tissue Samples ◽  
Response To Chemotherapy

Abstract Brain metastases (BM) are the most common neoplasm to affect the adult central nervous system, occurring ten times more frequently than primary brain tumors. BM develop in 40–50% of advanced lung adenocarcinoma (LUAD), but the lack of durable response to chemotherapy, immunotherapy, or targeted therapy results in death within a year of BM diagnosis. Several advances have been made in identifying genetic drivers of primary cancers. For instance, the ERK pathway is critical in oncogenesis, with aberrations linked to driving the progression and metastasis of LUAD. Unfortunately, BM remain poorly understood due to both the difficulty of obtaining a sizable collection of metastatic tissue samples as well as a lack of clinically relevant models. Typical models of BM utilize inoculation routes that are invasive and cannot fully recapitulate the entirety of the metastatic cascade. Here we present a novel murine model of BM through an intrathoracic inoculation method that recapitulates the development of primary lung tumors and brain metastases as seen in patients. Using this method, we evaluated the efficacy of LY3214996, an ERK inhibitor, in targeting BM. Seven days post-inoculation of a patient-derived NSCLC-BM line, mice received LY3214996 P.O. daily for 21 days, and were monitored weekly for metastases with bioluminescent imaging. Upon endpoint lungs and brains were also removed and imaged. In vitro we demonstrated a reduction in cell growth and invasion across multiple cell lines of varying KRAS status (NSCLC and NSCLC-BM) treated with LY3214996. In vivo, we saw a reduction in both lung tumor weight as well as frequency of BM. Further research is necessary to elucidate the dosing and efficacy of LY3214996 in our models in different genetic contexts. Nonetheless, our technique presents a novel preclinical tool to interrogate the metastatic process, allowing validation of genetic drivers as well as therapeutic screening.

scholarly journals TMOD-24. DEVELOPMENT OF A NOVEL IN VIVO MODEL OF LUNG-TO-BRAIN METASTASIS PROGRESSION

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi267-vi268
Author(s):  
Mohini Singh ◽  
Naema Nayyar ◽  
Megha Subramanian ◽  
Priscilla Brastianos
Keyword(s):  
Brain Metastases ◽  
Tumor Formation ◽  
In Vivo Model ◽  
Post Inoculation ◽  
Durable Response ◽  
Tissue Samples ◽  
Tumor Cell Suspension ◽  
Dismal Prognosis ◽  
Response To Chemotherapy

Abstract Brain metastases (BM) are the most common neoplasm to affect the adult central nervous system, occurring ten times more frequently than primary brain tumor. BM develop in 40–50% of advanced lung adenocarcinoma (LUAD), and the lack of durable response to chemotherapy, immunotherapy, or targeted therapy will result in death within a year of BM diagnosis. Despite the high burden of disease, dismal prognosis, and the increase in incidence over time, the biological underpinnings of BM remain poorly understood due to both the difficulty of obtaining a sizable collection of metastatic tissue samples as well as a lack of clinically relevant models. As such, it is possible that the inability to properly study this disease may result in metastasis driver-genes remaining undiscovered. Typical models of BM utilize direct implantation of tumor cells into the mouse brain, or inoculation into the blood via intracardiac/intracarotid injections; these routes are invasive and cannot fully recapitulate the entirety of the metastatic cascade. Here we present a novel, non-invasive method to develop primary lung tumors and brain metastases. A 10µL tumor cell suspension in phosphate-buffered saline was applied to the nostrils of lightly anesthetized mice, allowing direct deposit of cells into the lungs. Mice were monitored with bioluminescence imaging bi-weekly and culled at 2.5–3 months post-inoculation. Lungs and brains were also removed and imaged, where tumors in both organs were observed. Further research is necessary to elucidate either the linear or parallel progression of tumor formation within this model. Nonetheless, our technique presents a novel preclinical tool to interrogate the metastatic process, allowing validation of genetic drivers as well as therapeutic screening.

DDRE-03. THERAPEUTIC TARGETING OF THE ERK AND CDK PATHWAYS IN PRECLINICAL MODELS OF BRAIN METASTASES

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi74-vi75
Author(s):  
Mohini Singh ◽  
Naema Nayyar ◽  
Ashish Dahal ◽  
Priscilla Brastianos
Keyword(s):  
Cell Cycle ◽  
Brain Metastases ◽  
Cell Lines ◽  
Combined Treatment ◽  
Durable Response ◽  
Cdkn2a Mutation ◽  
Response To Chemotherapy ◽  
Cell Arrest

Abstract Brain metastases (BM) are the most common neoplasm to affect the adult central nervous system. BM develop in 40-50% of advanced lung adenocarcinoma (LUAD), but the lack of durable response to chemotherapy, immunotherapy, or targeted therapy results in death within a year of BM diagnosis. Several advances have been made in identifying genetic drivers of primary cancers. The cell cycle, RAS and ERK pathways have all been implicated in as critical oncogenic regulators, with aberrations linked to driving the progression and metastasis of LUAD. Abemaciclib is a targeted CDK4/6 inhibitor, and LY3214996 is selective ERK1/2 inhibitor, and have shown efficacy in preclinical tumor models as well as in clinical trials. Furthermore, both therapeutics can interfere with the cell cycle, abemaciclib through targeting CDK4/6 and LY3214996 through cyclinD1. Here we present data assessing abemaciclib and LY3214996, as single and combined agents, in cell lines across different KRAS and CDKN2A mutational backgrounds. Seven days post-intracranial inoculation of NSCLC and NSCLC-BM line, mice received either abemaciclib, LY3214996, or a combination P.O. daily for 21 days, and were monitored pre- and post-treatment for tumor growth with bioluminescent imaging. In vitro we demonstrated a dose-dependent reduction in cell growth with each treatment, as well as cell arrest in G1 phase. In vivo, whereas cell lines with a combined KRAS mutation and CDKN2A mutation/deletion had no significant reduction in BM growth, cell lines with a CDKN2A del or BRAF mutation had significant BM reduction, with single agents and combined treatment. Further research is necessary to elucidate under what genetic contexts abemaciclib, LY3214996 or the combination are most effective. Nonetheless, this work highlights that abemaciclib and LY3214996 should be further explored for CDKN2A or BRAF mutant BM.

scholarly journals Verification of 5-Aminolevurinic Radiodynamic Therapy Using a Murine Melanoma Brain Metastasis Model

2019 ◽  
Vol 20 (20) ◽  
pp. 5155 ◽  
Author(s):  
Junko Takahashi ◽  
Shinsuke Nagasawa ◽  
Mitsushi J. Ikemoto ◽  
Chikara Sato ◽  
Mari Sato ◽  
...  
Keyword(s):  
Brain Metastasis ◽  
Brain Metastases ◽  
Therapeutic Efficacy ◽  
Checkpoint Inhibitors ◽  
Melanoma Metastasis ◽  
X Rays ◽  
Melanoma Brain Metastasis ◽  
Metastasis Model

Melanoma is a highly aggressive cancer with a propensity for brain metastases. These can be treated by radiotherapy, but the radiation-resistant nature of melanoma makes the prognosis for melanoma patients with brain metastases poor. Previously, we demonstrated that treatment of mice with subcutaneous melanoma with 5-aminolevurinic acid (5-ALA) and X-rays in combination, (“radiodynamic therapy (RDT)”), instead of with 5-ALA and laser beams (“photodynamic therapy”), improved tumor suppression in vivo. Here, using the B16-Luc melanoma brain metastasis model, we demonstrate that 5-ALA RDT effectively treats brain metastasis. We also studied how 5-ALA RDT damages cells in vitro using a B16 melanoma culture. Cell culture preincubated with 5-ALA alone increased intracellular photosensitizer protoporphyrin IX. On X-ray irradiation, the cells enhanced their ∙OH radical generation, which subsequently induced γH2AX, a marker of DNA double-strand breaks in their nuclei, but decreased mitochondrial membrane potential. After two days, the cell cycle was arrested. When 5-ALA RDT was applied to the brain melanoma metastasis model in vivo, suppression of tumor growth was indicated. Therapeutic efficacy in melanoma treatment has recently been improved by molecular targeted drugs and immune checkpoint inhibitors. Treatment with these drugs is now expected to be combined with 5-ALA RDT to further improve therapeutic efficacy.

scholarly journals Establishment of an Acanthamoeba keratitis mouse model confirmed by amoebic DNA amplification

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Heekyoung Kang ◽  
Hae-Jin Sohn ◽  
A-Young Park ◽  
A-Jeong Ham ◽  
Jeong-Heon Lee ◽  
...  
Keyword(s):  
Mouse Model ◽  
Contact Lens ◽  
Dna Amplification ◽  
Ocular Tissue ◽  
In Vivo Model ◽  
Rrna Gene ◽  
Post Inoculation ◽  
Therapeutic Drugs

AbstractAcanthamoeba castellanii, the causative agent of Acanthamoeba keratitis (AK), occurs mainly in contact lens users with poor eye hygiene. The findings of many in vitro studies of AK, as well as the testing of therapeutic drugs, need validation in in vivo experiments. BALB/c mice were used in this study to establish in vivo AK model. A. castellanii cell suspensions (equal mixtures of trophozoites and cysts) were loaded onto 2-mm contact lens pieces and inserted into mouse eyes that were scratched using an ophthalmic surgical blade under anesthesia and the eyelids of the mice were sutured. The AK signs were grossly observed and PCR was performed using P-FLA primers to amplify the Acanthamoeba 18S-rRNA gene from mouse ocular tissue. The experimental AK mouse model was characterized by typical hazy blurring and melting of the mouse cornea established on day 1 post-inoculation. AK was induced with at least 0.3 × 105A. castellanii cells (optimal number, 5 × 104), and the infection persisted for two months. The PCR products amplified from the extracted mouse eye DNA confirmed the development of Acanthamoeba-induced keratitis during the infection periods. In conclusion, the present AK mouse model may serve as an important in vivo model for the development of various therapeutic drugs against AK.

scholarly journals Prophylactic TLR9 stimulation reduces brain metastasis through microglia activation

2019 ◽  
Author(s):  
Amit Benbenishty ◽  
Meital Gadrich ◽  
Azzurra Cottarelli ◽  
Alisa Lubart ◽  
David Kain ◽  
...  
Keyword(s):  
Brain Metastasis ◽  
Brain Metastases ◽  
Perioperative Period ◽  
Barrier Integrity ◽  
Activated Microglia ◽  
Tumor Killing ◽  
The Brain ◽  
Blood Brain Barrier Integrity

AbstractBrain metastases are prevalent in various types of cancer, and are often terminal given low efficacy of available therapies. Therefore, preventing them is of outmost clinical relevance and prophylactic treatments are perhaps the most efficient strategy. Here, we show that systemic prophylactic administration of a TLR9 agonist, CpG-C, is effective against brain metastases. Acute and chronic systemic administration of CpG-C reduced tumor cell seeding and growth in the brain in three tumor models in mice, including metastasis of human and mouse lung cancer, and spontaneous melanoma-derived brain metastasis. Studying mechanisms underlying the therapeutic effects of CpG-C, we found that in the brain, unlike in the periphery, NK cells and monocytes are not involved in controlling metastasis. Next, we demonstrated that the systemically administered CpG-C is taken up by endothelial cells, astrocytes, and microglia, without affecting blood-brain barrier integrity and tumor brain extravasation. In vitro assays pointed to microglia, but not astrocytes, as mediators of CpG-C effects through increased tumor killing and phagocytosis, mediated by direct microglia-tumor contact. In vivo, CpG-C-activated microglia displayed elevated mRNA expression levels of apoptosis-inducing and phagocytosis-related genes. Intravital imaging showed that CpG-C-activated microglia cells contact, kill, and phagocytize tumor cells in the early stages of tumor brain invasion more than non-activated microglia. Blocking in vivo activation of microglia with minocycline, and depletion of microglia with a colony-stimulating factor 1 inhibitor, indicated that microglia mediate the anti-tumor effects of CpG-C. Overall, the results suggest prophylactic CpG-C treatment as a new intervention against brain metastasis, through an essential activation of microglia.SummaryBrain metastases are prevalent and often terminal. Thus, reducing their occurrence could markedly improve cancer outcome. We show that systemic prophylactic and perioperative administration of a TLR9 agonist, CpG-C, reduced metastatic growth in experimental and spontaneous brain metastasis models, employing mouse and human tumors. CpG-C was taken up in the brain, without affecting blood-brain barrier integrity and tumor extravasation. In vitro assays, imaging flow cytometry, and intravital imaging pointed to microglia as mediators of CpG-C effects through contact-dependent tumor killing and phagocytosis; corresponding with in vivo mRNA profile. In vivo depletion studies proved that microglia, but not NK cells or monocytes, mediated the beneficial effects of CpG-C; Also hindered by blocking microglial activation. In-toto, perioperative treatment with CpG-C should be considered clinically relevant.SignificancePreventing brain metastases is paramount, as they are considered incurable and their incidence is on the rise due to prolonged survival of cancer patients. Here, we demonstrate that systemic prophylactic treatment with CpG-C reduces peripheral and brain metastasis of mouse and human lung cancers. While traditional therapies are halted during the perioperative period, we found systemic CpG-C treatment during this time frame beneficial in a model of spontaneous brain metastases following excision of a primary melanoma tumor, comprehensively mimicking the clinical setting. Mechanistically, we show microglia activation with CpG-C results in tumor cell eradication, pointing to microglia as potential therapeutic targets. Importantly, CpG-ODNs have negligible toxicity in humans. Therefore, CpG-C may be used prophylactically and during the perioperative period in high-risk cancers.

scholarly journals STEM-23. METASTATIC BRAIN TUMOR IMAGING THROUGH TARGETED PEPTIDES ISOLATED VIA PHAGE DISPLAY BIOPANNING AGAINST BRAIN METASTASIS-INITIATING CELLS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi238-vi238
Author(s):  
JongMyung Kim ◽  
James Liu
Keyword(s):  
Lung Cancer ◽  
Brain Metastasis ◽  
Brain Metastases ◽  
Phage Display ◽  
Tumor Imaging ◽  
Mesenchymal Transition ◽  
Phage Display Biopanning ◽  
In Vivo Phage Display

Abstract To effectively target metastatic brain tumors (MBTs), the paradigm of initiating treatment against MBTs following detection on clinical imaging needs to be shifted to an understanding of the mechanisms that drive the formation and maintenance of brain metastasis-initiating cells (BMICs). Targeting this tumor sub-population, which may form as a result of activation of the epithelial-mesenchymal transition, may allow for more effective means of understanding and targeting brain metastases. In order to isolate BMICs, we have harvested cells from patient derived MBTs originating from lung cancer and cultured the cells using stem cell media conditions. We then performed in vitro and in vivo phage display biopanning to isolate 12-amino acid length peptides that specifically target BMICs. Several peptides were isolated from both in vitro and in vivo biopanning strategies. Of the peptides recovered, one peptide, LBM4, demonstrated specific binding to MBT cells over primary lung cancer cells in vitro through flow cytometry analysis and immunocytochemistry. Fluorescent tagged LBM4 intravenously injected into mice harboring intracranial brain metastases demonstrated peptide localization to the tumor within the intracranial cavity visualized with live animal imaging. Peptide imaging of tumor corresponded to MRI imaging confirming that the peptides could serve as an alternative to tumor imaging, with the potential for greater sensitivity resulting from the cellular targeting of MBTs. Our results demonstrate that we can use a combination of in vitro and in vivo phage display biopanning to isolate cell specific targeting peptides. MBT targeting peptides can potentially result in a shifting of the clinical treatment paradigm of brain metastases, through the development of more effective targeted therapeutics aimed at BMICs, as well as improving detection of MBT cells which may result in earlier tumor visualization, as well as delineation of tumor recurrence versus radiation effects.

scholarly journals TAMI-03. IDENTIFICATION OF NOVEL DRIVERS OF LUNG-TO-BRAIN METASTASIS THROUGH IN VIVO FUNCTIONAL GENOMICS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii213-ii213
Author(s):  
Nikoo Aghaei ◽  
Fred Lam ◽  
Chitra Venugopal ◽  
Sheila Singh
Keyword(s):  
Brain Metastasis ◽  
Brain Metastases ◽  
Lung Tumor ◽  
Unmet Need ◽  
Xenograft Model ◽  
Tumor Formation ◽  
Loss Of Function ◽  
Mri Imaging ◽  
Genome Wide

Abstract Brain metastasis, the most common tumor of the central nervous system, occurs in 20-36% of primary cancers. In particular, 40% of patients with non-small cell lung cancer (NSCLC) develop brain metastases, with a dismal survival of approximately 4-11 weeks without treatment, and 16 months with treatment. This highlights a large unmet need to develop novel targeted therapies for the treatment of lung-to-brain metastases (LBM). Genomic interrogation of LBM using CRISPR technology can inform preventative therapies targeting genetic vulnerabilities in both primary and metastatic tumors. Loss-of-function studies present limitations in metastasis research, as knocking out genes essential for survival in the primary tumor cells can thwart the metastatic cascade prematurely. However, gene overexpression using CRISPR activation (CRISPRa) has the potential for overcoming dependencies of gene essentiality. We theorize that an in vivo genome-wide CRISPRa screen will identify novel genes that, when overexpressed, drive LBM. We have developed a patient-derived orthotopic murine xenograft model of LBM using primary patient-derived NSCLC cell lines (termed LTX cells) from the Swanton Lab TRACERx study. We are now poised to transduce LTX cells with a human genome-wide CRISPRa single guide RNA (sgRNA) library, and to subsequently inject the cells into the lungs of immunocompromised mice. We will then track the process of LBM using bioluminescent and MRI imaging until mice reach endpoint. Sequencing of primary lung tumors and subsequent brain metastases promises to uncover enriched sgRNAs, which may represent novel drivers of primary lung tumor formation and LBM. To the best of our knowledge, this study is the first in vivo genome-wide CRISPRa screen focused on identifying novel drivers of LBM, and can inform future preventative therapies to improve survival outcomes for NSCLC patients.

Tamarix articulata (T. articulata) - An Important Halophytic Medicinal Plant with Potential Pharmacological Properties

2019 ◽  
Vol 20 (4) ◽  
pp. 285-292 ◽  
Author(s):  
Abdullah M. Alnuqaydan ◽  
Bilal Rah
Keyword(s):  
Medicinal Plant ◽  
Biological Activities ◽  
Wide Spectrum ◽  
Biological Properties ◽  
In Vivo Model ◽  
Drug Candidate ◽  
Phytochemical Analysis ◽  
Pharmacological Properties

Background:Tamarix Articulata (T. articulata), commonly known as Tamarisk or Athal in Arabic region, belongs to the Tamaricaece species. It is an important halophytic medicinal plant and a good source of polyphenolic phytochemical(s). In traditional medicines, T. articulata extract is commonly used, either singly or in combination with other plant extracts against different ailments since ancient times.Methods:Electronic database survey via Pubmed, Google Scholar, Researchgate, Scopus and Science Direct were used to review the scientific inputs until October 2018, by searching appropriate keywords. Literature related to pharmacological activities of T. articulata, Tamarix species, phytochemical analysis of T. articulata, biological activities of T. articulata extracts. All of these terms were used to search the scientific literature associated with T. articulata; the dosage of extract, route of administration, extract type, and in-vitro and in-vivo model.Results:Numerous reports revealed that T. articulata contains a wide spectrum of phytochemical(s), which enables it to have a wide window of biological properties. Owing to the presence of high content of phytochemical compounds like polyphenolics and flavonoids, T. articulata is a potential source of antioxidant, anti-inflammatory and antiproliferative properties. In view of these pharmacological properties, T. articulata could be a potential drug candidate to treat various clinical conditions including cancer in the near future.Conclusion:In this review, the spectrum of phytochemical(s) has been summarized for their pharmacological properties and the mechanisms of action, and the possible potential therapeutic applications of this plant against various diseases discussed.

scholarly journals Radiofrequency Irradiation Modulates TRPV1-Related Burning Sensation in Rosacea

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1424
Author(s):  
Seyeon Oh ◽  
Myeongjoo Son ◽  
Joonhong Park ◽  
Donghwan Kang ◽  
Kyunghee Byun
Keyword(s):  
Burning Sensation ◽  
In Vivo Model ◽  
Molecular Evidence ◽  
Exposed Skin ◽  
Heat Treated ◽  
Vitro Model ◽  
Effective Use ◽  
Inflammatory Molecules

Rosacea is a skin inflammatory condition that is accompanied by not only redness and flushing but also unseen symptoms, such as burning, stinging, and itching. TRPV1 expression in UVB-exposed skin can lead to a painful burning sensation. Upregulated TRPV1 expression helps release neuropeptides, including calcitonin gene-related peptide, pituitary adenylate cyclase-activating polypeptide, and vasoactive intestinal peptide, which can activate macrophage and inflammatory molecules. In this study, we found that radiofrequency (RF) irradiation reduced TRPV1 activation and neuropeptide expression in a UVB-exposed in vivo model and UVB- or heat-treated in an in vitro model. RF irradiation attenuated neuropeptide-induced macrophage activation and inflammatory molecule expression. Interestingly, the burning sensation in the skin of UVB-exposed mice and patients with rosacea was significantly decreased by RF irradiation. These results can provide experimental and molecular evidence on the effective use of RF irradiation for the burning sensation in patients with rosacea.

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