scholarly journals TMOD-02. IN VITRO & IN VIVO CULTURE OF PATIENT DERIVED (PD) CSF-CTCS IN LEPTOMENINGEAL DISEASE (LMD) FROM MELANOMA TO IDENTIFY NOVEL TREATMENT STRATEGIES

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii227-ii228
Author(s):  
Vincent Law ◽  
Brittany Evernden ◽  
John Puskas ◽  
Gisela Caceres ◽  
Elena Ryzhova ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Treatment Strategies ◽  
Mek Inhibitor ◽  
Leptomeningeal Disease ◽  
Therapeutic Effects ◽  
In Vivo Models ◽  
In Vivo Culture ◽  
In Vivo Testing

Abstract BACKGROUND Approx. 5% of melanoma pts develop LMD. There are essentially no models of LMD available for therapeutic development. A significant barrier to the development of effective therapies against LMD has been the inability to culture and expand LMD cells. Here we report our strategies to in vitro & in vivo culturing of CSF-CTCs. As a proof of concept, we assessed response to Ceritinib (Cer), a non-canonical IGF1R inhibitor) in combination with MEK inhibitor. METHODS We collected CSF from 11 patients (pts) from various sources (ie: LPs, Ommayas, autopsies). 3 pts CSF were collected at autopsies. PD-CSF-CTCs were expanded in vitro in conditioned media and in vivo using CDX model. scRNAseq analysis was performed to assess expression profiles of PD-CSF-CTCs. RESULTS AND DISCUSSION Of the total 61 PD-CSF-CTCs collected from 11 pts (avg: 4.07 CSF collections/patient), we successfully cultured PD-CSF-CTCs from 3 pts (20%) and were able to grow them in vivo from 2 pts (18%). scRNAseq analysis identified MLANA, IGF1R, SOX9 and ErbB3 were among genes highly expressed in our PD-CSF-CTCs. We evaluated the responses of the combination Cer with MEKi (Tra) in vitro and in vivo and found that these agents produced therapeutic effects to both established melanoma cell lines and our PD-CSF-CTCs. For example, in vivo testing showed a median survival (MS): 18, 35, and 27 days in WM164, WM164R and the PD-CSF-CTCs, respectively, in control groups. Whereas treatment with Cer + Tra produced significantly better MS in all three in vivo models and was not reached in WM164, WM164R (p< 0.001 & p< 0.047, respectively) and 38.5 days in PD-CSF-CTCs (p< 0.032). CONCLUSIONS Though the sample size is small, this is the first report of the successful in vitro & in vivo culture of CSF-CTCs from pts with LMD.

scholarly journals LMD-07. In Vitro AND In Vivo Culture of Patient Derived-Cerebral Spinal Fluid-Circulating Tumor Cells (PD-CSF-CTCs) in Leptomeningeal Disease (LMD) From Melanoma to Identify Novel Treatment Strategies

2021 ◽  
Vol 3 (Supplement_3) ◽  
pp. iii8-iii8
Author(s):  
Vincent Law ◽  
Zhihua Chen ◽  
Inna Smalley ◽  
Francesca Vena ◽  
Robert Macaulay ◽  
...  
Keyword(s):  
Cell Line ◽  
Treatment Strategies ◽  
Xenograft Model ◽  
Braf Inhibitor ◽  
Mek Inhibitor ◽  
P Value ◽  
Leptomeningeal Disease ◽  
In Vivo Culture

Abstract Background Approximately 5% of melanoma patients (pts) will develop LMD. Currently there is no effective treatments for this disease. A significant barrier to the development of effective therapies has been the inability to culture CSF-CTCs for functional analysis. For the first time, we were able to successfully expand CSF-CTCs in vitro and in vivo. We assessed gene signatures of PD-CSF-CTCs to determine novel targets for therapy. As a proof of concept, we tested the efficacy of combining ceritinib (cer), an IGF-1R inhibitor and trametinib (tra), a MEK inhibitor, against LMD. Methods CSF from 11 pts were collected from various sources (ie: LPs, Ommayas, rapid autopsies). PD-CSF-CTCs were expanded in vitro in conditioned media and in vivo using cell line-derived xenograft model. Single-cell RNA-sequencing (scRNAseq) analysis was performed to assess transcriptional profiles of PD-CSF-CTCs. Results Of the total 61 PD-CSF-CTCs collected from 11 pts (avg: 4.07 CSF collections/patient), we successfully cultured PD-CSF-CTCs from 3 pts (20%) and were able to grow them in vivo from 2 pts (18%). scRNAseq identified IGF-1R, Sox9, ErbB3 and MLANA were among the enriched genes for PD-CSF-CTCs. IGF-1R inhibition by cer and depletion by CRISPR suppressed cell growth. We evaluated the responses of cer + tra treatment in vitro and found that combining these agents produced drug synergy against PD-CSF-CTCs and resensitized BRAF inhibitor-resistant melanoma cell line, WM164R. In vivo LMD xenograft model showed cer + tra treatment significantly prolonged median survival of PD-CSF-CTCs LMD (control: 27 days vs treatment: 38.5 days; P value < 0.032) and WM164R LMD (control: 35 days vs treatment: MS not reached; P value < 0.047). Conclusions Though the sample size is small, this is the first report of the successful in vitro and in vivo culture of CSF-CTCs from pts with LMD.

scholarly journals CMET-26. IN-VITRO & IN-VIVO CULTURE OF PATIENT (PT) DERIVED CSF-CTCS IN LEPTOMENINGEAL DISEASE (LMDZ) FROM MELANOMA TO IDENTIFY NOVEL TREATMENT STRATEGIES

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi57-vi57
Author(s):  
Vincent Law ◽  
Brittany Evernden ◽  
John Puskas ◽  
Gisela Caceres ◽  
Elena Ryzhova ◽  
...  
Keyword(s):  
Single Cell Analysis ◽  
Ex Vivo ◽  
Treatment Strategies ◽  
Braf V600e ◽  
Leptomeningeal Disease ◽  
Immunodeficient Mice ◽  
In Vivo Culture ◽  
In Vivo Testing

Abstract BACKGROUND Approx. 5% of melanoma pts develop LMDz. There are essentially no models of LMDz available for therapeutic development. Here we report, the in-vitro & in-vivo culturing of CSF-CTCs. METHODS CSF-CTCs were detected by the Veridex CellSearch® System. Cell-free DNA and cell-associated DNA were extracted, sequenced and profiled. Expanded ex-vivo CSF-CTCs were grown in-vitro and tested for drug sensitivity. CSF-CTCs were grown successfully in-vivo from 1 pt; labeled human Braf V600E WM164 cells were injected IT in as a control. RESULTS CSF-CTCs: 12 LMDz pts and 8 melanoma pts without LMDz were studied. All but 1 LMDz pts (92%) had CSF-CTCs (avg: 2148.6; range 23 - 3055 CTCs/ml). In contrast, 3/8 (37%) melanoma Brain Mets pts without LMDz had CSF-CTCs but fewer of them (avg: 0.31; range 0.13 - 0.6 CTCs/ml CSF). CSF-CTCs Profile: These had BrafV600E (83%), and GNAQ Q209P & NRAS Q61R in 1 pt each. Ex-vivoculture of CSF-CTCs and PDX model: After lengthy optimization of conditions we successfully expanded CSF-CTCs in vitro(~25% of pts), and in-vivo in immunodeficient mice from 1 pt (~10% of samples). Ceritinib, used as a FAK inhibitor, with MEKi was effective in-vitro (p=3.17e-6) and prolonged survival in-vivo in LMDz (median survival: >32 days vs control: 18 days; p=7.81e-5). CONCLUSIONS Though the sample size is small, this is the first report of the successful in-vitro & in-vivo culture of CSF-CTCs from pts with LMDz. Single cell analysis to determine how representative these models are and further in-vivo testing are in progress.

scholarly journals LPTO-03. IN-VITRO & IN-VIVO CULTURE OF PATIENT (PT) DERIVED CSF-CTCs IN LEPTOMENINGEAL DISEASE (LMDz) FROM MELANOMA TO IDENTIFY NOVEL TREATMENT STRATEGIES

2019 ◽  
Vol 1 (Supplement_1) ◽  
pp. i6-i7
Author(s):  
Vincent Law ◽  
Brittany Evernden ◽  
Rajappa Kenchappa ◽  
John Puskas ◽  
Gisela Caceres ◽  
...  
Keyword(s):  
Single Cell Analysis ◽  
Ex Vivo ◽  
Treatment Strategies ◽  
Braf V600e ◽  
Leptomeningeal Disease ◽  
Immunodeficient Mice ◽  
In Vivo Culture ◽  
In Vivo Testing

Abstract BACKGROUND: Approximately 5% of melanoma pts develop LMDz. There are essentially no models of LMDz available for therapeutic development. Here we report, the in-vitro & in-vivo culturing of CSF-CTCs. METHODS: CSF-CTCs were detected by the Veridex CellSearch® System. Cell-free DNA and cell-associated DNA were extracted, sequenced and profiled. Expanded ex-vivo CSF-CTCs were grown in-vitro and tested for drug sensitivity. CSF-CTCs were grown successfully in-vivo from 1 pt; labeled human Braf V600E WM164 cells were injected IT in as a control. RESULTS: CSF-CTCs: 12 LMDz pts and 8 melanoma pts without LMDz were studied. All but 1 LMDz pts (92%) had CSF-CTCs (avg: 2148.60; range 23 - 3055 CTCs/ml). In contrast, 3/8 (37%) melanoma Brain Mets pts without LMDz had CSF-CTCs but fewer of them (avg: 0.31; range 0.13 - 0.6 CTCs/ml CSF). CSF-CTCs Profile: These had BrafV600E (83%), and GNAQ Q209P & NRAS Q61R in 1 pt each. Ex-vivo culture of CSF-CTCs and PDX model: After lengthy optimization of conditions we successfully expanded CSF-CTCs in-vitro (~25% of pts), and in-vivo in immunodeficient mice from 1 pt (~10% of samples). Ceritinib, used as a FAK inhibitor, with MEKi was effective in-vitro (p=3.17e-6) and prolonged survival in-vivo in LMDz (median survival: >32 days vs control: 18 days; p=7.81e-5). CONCLUSIONS: Though the sample size is small, this is the first report of the successful in-vitro & in-vivo culture of CSF-CTCs from pts with LMDz. Single cell analysis to determine how representative these models are and further in-vivo testing are in progress.

scholarly journals DDEL-12. NANOPARTICLE DELIVERY OF DOXORUBICIN FOR THE TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii286-iii286
Author(s):  
Caitlin Ung ◽  
Maria Tsoli ◽  
Jie Liu ◽  
Domenico Cassano ◽  
Dannielle Upton ◽  
...  
Keyword(s):  
Treatment Strategies ◽  
Pediatric Brain Tumors ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Confocal Imaging ◽  
Cell Content ◽  
Potent Effect ◽  
Orthotopic Mouse Model ◽  
In Vivo Testing

Abstract DIPGs are the most aggressive pediatric brain tumors. Currently, the only treatment is irradiation but due to its palliative nature patients die within 12 months. Effective delivery of chemotherapy across the blood-brain barrier (BBB) has been a key challenge for the eradication of this disease. We have developed a novel gold nanoparticle functionalised with human serum albumin (Au-NP, 98.8 ±19 nm) for the delivery of doxorubicin. In this study, we evaluated the cytotoxic efficacy of doxorubicin delivered through gold nanoparticles (Au-NP-Dox). We found that DIPG neurospheres were equally sensitive to doxorubicin and Au-NP-Dox (at equimolar concentration) by alamar blue assay. Colony formation assays demonstrated a significantly more potent effect of Au-NP-Dox compared to doxorubicin alone, while the Au-NP had no effect. Furthermore, western blot analysis indicated increased apoptotic markers cleaved Parp, caspase 3/7 and phosphorylated H2AX in Au-NP-Dox treated DIPG neurospheres. Live cell content and confocal imaging demonstrated significantly higher uptake of Au-NP-Dox compared to doxorubicin alone. Treatment of a DIPG orthotopic mouse model with Au-NP-Dox showed no signs of toxicity with stable weights being maintained during treatment. However, in contrast to the above in vitro findings the in vivo study showed no anti-tumor effect possibly due to poor penetration of Au-NP-Dox into the brain. We are currently evaluating whether efficacy can be improved using measures to open the BBB transiently. This study highlights the need for rigorous in vivo testing of new treatment strategies before clinical translation to reduce the risk of administration of ineffective treatments.

scholarly journals In Vitro and In Vivo Models of Cerebral Ischemia Show Discrepancy in Therapeutic Effects of M2 Macrophages

PLoS ONE ◽  
2013 ◽  
Vol 8 (6) ◽  
pp. e67063 ◽  
Author(s):  
Virginie Desestret ◽  
Adrien Riou ◽  
Fabien Chauveau ◽  
Tae-Hee Cho ◽  
Emilie Devillard ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Therapeutic Effects ◽  
M2 Macrophages ◽  
In Vivo Models

220 THE ESTROGEN-LIKE EFFECT OF 2-METHOXYESTRADIOL (2-ME) IN IN VITRO AND IN VIVO MODELS

2015 ◽  
Vol 27 (1) ◽  
pp. 200
Author(s):  
J.-S. Lee ◽  
E.-B. Jeung
Keyword(s):  
Mrna Expression ◽  
Therapeutic Effects ◽  
Mrna Levels ◽  
Oestrogen Receptors ◽  
In Vivo Models ◽  
Ru 486 ◽  
Final Injection ◽  
M 10

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17β-oestradiol, interacts with oestrogen receptors and microtubules and has a low affinity for oestrogen receptors (ER). It has attracted considerable interest due to its potential anti-cancer therapeutic effects. 2-ME is also recognised for its unique and profound actions on various tumour cell lines and cancer independent of the hormone receptor status. Regardless of differences in function, 2-ME has an affinity for ER, however, the exact mechanisms of 2-ME action via the ER are not fully understood. In the current study, we examined the estrogenic effect of 2-ME on mRNA levels of CaBP-9k, ER, and progesterone receptor (PR) in the absence or presence of the 17β-oestradiol (E2) and progesterone (P4) in both in vivo and in vitro models by real-time RT–PCR. In vitro, cells (n = 3 per group) were exposed to a single dose of E2 (10–9 M), P4 (10–6 M), 2-ME (10–8 M, 10–7 M, 10–6 M). The mechanism of CaBP-9k induction by these chemicals pre-treated with 10–7 M ICI 182, 780 and 10–6 M RU 486 for 30 min before exposure to E2 and 2-ME were analysed. In vivo, 35 female ICR mice (PND 14 days) were divided into 7 groups (n = 5 per group), and each group was administered subcutaneously with 24% DMSO, 38% ethanol, and 38% sterile saline as a vehicle, E2 [40 μg kg–1 of body weight (BW)] a physiological dose level), 2-ME (4, 40, and 80 mg kg–1 of BW) for 3 days. The mice were killed 24 h after the final injection. To investigate the effect of antagonism, 10 mice were injected SC with ICI 182 780 (10 mg kg–1 of BW) and RU 486 (10 mg kg–1 of BW) at 30 min before injection with 2-ME (40 mg kg–1 of BW) for 3 days and killed 24 h after the final injection. Results are presented as mean ± s.e.m.; P-values were calculated using one-way ANOVA. In GH3 cells, the mRNA level of CaBP-9k was induced in the E2 (10–9 M) treatment group, and expression of CaBP-9k was also up-regulated in the 2-ME (10–7 M)-treated group. Uterine lactoferrin (Ltf) mRNA expression was also increased in the 2-ME (40 mg kg–1 of BW) group, similar to the response with E2 (40 μg kg–1 of BW) in mice. As a blocker for ER and PR activity, ICI 182 780 and RU 486 reversed the E2 or 2-ME mediated increase of CaBP-9k and Ltf mRNA expression. We found that 2-ME significantly increased the levels of ERa and PR transcripts. In parallel with in vitro results, the mRNA levels of ERa and PR were induced by treatment with E2 and 2-ME. Taken together, our findings demonstrated that expression of estrogenic markers, CaBP-9k and Ltf, was regulated by 2-ME in both in vitro and in vivo, which may increase their estrogenic activities in female during the cycle through ER and/or PR-mediated pathway.

scholarly journals Colorectal Cancer Growth Retardation through Induction of Apoptosis, Using an Optimized Synergistic Cocktail of Axitinib, Erlotinib, and Dasatinib

Cancers ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1878 ◽  
Author(s):  
Robert H. Berndsen ◽  
Nathalie Swier ◽  
Judy R. van Beijnum ◽  
Patrycja Nowak-Sliwinska
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Expression Profiles ◽  
Chorioallantoic Membrane ◽  
Treatment Strategies ◽  
Tumor Growth Inhibition ◽  
Clinical Implementation

Patients with advanced colorectal cancer (CRC) still depend on chemotherapy regimens that are associated with significant limitations, including resistance and toxicity. The contribution of tyrosine kinase inhibitors (TKIs) to the prolongation of survival in these patients is limited, hampering clinical implementation. It is suggested that an optimal combination of appropriate TKIs can outperform treatment strategies that contain chemotherapy. We have previously identified a strongly synergistic drug combination (SDC), consisting of axitinib, erlotinib, and dasatinib that is active in renal cell carcinoma cells. In this study, we investigated the activity of this SDC in different CRC cell lines (SW620, HT29, and DLD-1) in more detail. SDC treatment significantly and synergistically decreased cell metabolic activity and induced apoptosis. The translation of the in-vitro-based results to in vivo conditions revealed significant CRC tumor growth inhibition, as evaluated in the chicken chorioallantoic membrane (CAM) model. Phosphoproteomics analysis of the tested cell lines revealed expression profiles that explained the observed activity. In conclusion, we demonstrate promising activity of an optimized mixture of axitinib, erlotinib, and dasatinib in CRC cells, and suggest further translational development of this drug mixture.

scholarly journals Therapeutic Effects of Curcumin—From Traditional Past to Present and Future Clinical Applications

2019 ◽  
Vol 20 (15) ◽  
pp. 3757 ◽  
Author(s):  
Beatrice Bachmeier ◽  
Dieter Melchart
Keyword(s):  
Molecular Mechanisms ◽  
Malignant Tumors ◽  
Scientific Evidence ◽  
Treatment Strategies ◽  
Therapeutic Effects ◽  
Microbial Infection ◽  
Therapeutic Benefits ◽  
Novel Treatment Strategies

The efficacy of the plant-derived polyphenol curcumin, in various aspects of health and wellbeing, is matter of public interest. An internet search of the term “Curcumin” displays about 12 million hits. Among the multitudinous information presented on partly doubtful websites, there are reports attracting the reader with promises ranging from eternal youth to cures for incurable diseases. Unfortunately, many of these reports are not based on scientific evidence, but they feed the desideratum of the reader for a “miracle cure”. This circumstance makes it very difficult for researchers, who work in a scientifically sound and evidence-based manner on the therapeutic benefits (or side effects) of curcumin, to demarcate their results from sensational reports that circulate in the web and in other media. This is only one of many obstacles making it difficult to pave curcumin’s way into clinical application; others are its nonpatentability and low economic usability. A further impediment comes from scientists who never worked with curcumin or any other natural plant-derived compound in their own labs. They have never tested these compounds in any scientific assay, neither in vitro nor in vivo; however, they claim, in a sometimes polemic manner, that everything that has so far been published on curcumin’s molecular effects is based on artefacts. The here presented Special Issue comprises a collection of five scientifically sound articles and nine reviews reporting on the therapeutic benefits and the molecular mechanisms of curcumin or of chemically modified curcumin in various diseases ranging from malignant tumors to chronic diseases, microbial infection, and even neurodegenerative diseases. The excellent results of the scientific projects that underlie the five original papers give reason to hope that curcumin will be part of novel treatment strategies in the near future—either as monotherapy or in combination with other drugs or therapeutic applications.

Somatic Mutational Landscape of AML with Inv(16) and t(8;21) Identifies Two Distinct Patterns in Relapse Tumors

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2362-2362
Author(s):  
Raman B. Sood ◽  
Nancy F Hansen ◽  
Frank X Donovan ◽  
Blake Carrington ◽  
Baishali Maskeri ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Clonal Evolution ◽  
Treatment Strategies ◽  
Gene Mutations ◽  
Driver Gene ◽  
Intrinsic Resistance ◽  
Driver Genes ◽  
In Vivo Models

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease with a wide prognostic spectrum ranging from poor to good depending upon the underlying mutations and/or cytogenetic abnormalities. Although AMLs with inv(16)/t(16:16) or t(8,21), collectively referred to as core binding factor leukemias (CBF-AMLs), are classified as prognostically favorable, such patients often succumb to their disease following relapse after an initial response to cytarabine/anthracyclin-based treatment regimens. Thus, to develop successful treatment strategies, it is critical to understand the mechanisms leading to disease relapse and target them with novel therapeutic approaches. To pursue this goal, we applied genomic approaches (whole exome sequencing and single nucleotide polymorphism arrays) on DNA from samples collected at sequential time points (i.e., diagnosis, complete remission and relapse) in seven patients with inv(16) and six patients with t(8;21). We identified mutations in several previously identified AML driver genes, such as KIT, FLT3, DNMT3A, EZH2, SMC1A, SMC3, WT1 and NRAS. Three relapse samples showed mosaicism for monosomy/disomy of the region of chromosome 3 containing GATA2. Overall, our data revealed two distinct profiles that support different mechanisms of relapse: 1) diagnosis and relapse blasts harbor the same driver gene mutations, indicating the intrinsic resistance of the major clones present at diagnosis to treatment regimen used; 2) diagnosis and relapse tumors have different driver gene mutations, indicating disease clonal evolution possibly through treatment selective pressure. Furthermore, our data has identified previously unreported putative driver genes for AML. Among these, we identified same somatic variant (R222G) in DHX15, an RNA helicase involved in splicing, in two patients at diagnosis. The variant was also detected at relapse in one of these patients. Functional validation of the mechanistic roles of wild type and mutated DHX15 in hematopoiesis and leukemogenesis, respectively, is ongoing in in vitro and in vivo models. Disclosures No relevant conflicts of interest to declare.

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