Examination of the Utility of the High Throughput In Vitro Metabolic Stability Assay to Estimate In Vivo Clearance in the Mouse

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

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O23: CHARACTERISING PATIENT-DERIVED COLORECTAL CANCER TISSUE-ORIGINATED ORGANOIDAL SPHEROIDS FOR HIGH-THROUGHPUT MICROFLUIDIC APPLICATIONS

British Journal of Surgery ◽

10.1093/bjs/znab117.023 ◽

2021 ◽

Vol 108 (Supplement_1) ◽

Author(s):

MI Khot ◽

M Levenstein ◽

R Coppo ◽

J Kondo ◽

M Inoue ◽

...

Keyword(s):

Colorectal Cancer ◽

Fluid Flow ◽

High Throughput ◽

Microfluidic Devices ◽

In Vitro Models ◽

Fluorescent Imaging ◽

Cancer Tissue ◽

Cell Models

Abstract Introduction Three-dimensional (3D) cell models have gained reputation as better representations of in vivo cancers as compared to monolayered cultures. Recently, patient tumour tissue-derived organoids have advanced the scope of complex in vitro models, by allowing patient-specific tumour cultures to be generated for developing new medicines and patient-tailored treatments. Integrating 3D cell and organoid culturing into microfluidics, can streamline traditional protocols and allow complex and precise high-throughput experiments to be performed with ease. Method Patient-derived colorectal cancer tissue-originated organoidal spheroids (CTOS) cultures were acquired from Kyoto University, Japan. CTOS were cultured in Matrigel and stem-cell media. CTOS were treated with 5-fluorouracil and cytotoxicity evaluated via fluorescent imaging and ATP assay. CTOS were embedded, sectioned and subjected to H&E staining and immunofluorescence for ABCG2 and Ki67 proteins. HT29 colorectal cancer spheroids were produced on microfluidic devices using cell suspensions and subjected to 5-fluorouracil treatment via fluid flow. Cytotoxicity was evaluated through fluorescent imaging and LDH assay. Result 5-fluorouracil dose-dependent reduction in cell viability was observed in CTOS cultures (p<0.01). Colorectal CTOS cultures retained the histology, tissue architecture and protein expression of the colonic epithelial structure. Uniform 3D HT29 spheroids were generated in the microfluidic devices. 5-fluorouracil treatment of spheroids and cytotoxic analysis was achieved conveniently through fluid flow. Conclusion Patient-derived CTOS are better complex models of in vivo cancers than 3D cell models and can improve the clinical translation of novel treatments. Microfluidics can streamline high-throughput screening and reduce the practical difficulties of conventional organoid and 3D cell culturing. Take-home message Organoids are the most advanced in vitro models of clinical cancers. Microfluidics can streamline and improve traditional laboratory experiments.

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ShearFAST: a user-friendly in vitro toolset for high throughput, inexpensive fluid shear stress experiments.

10.1101/2020.01.31.929513 ◽

2020 ◽

Author(s):

Thomas Brendan Smith ◽

Alessandro Marco De Nunzio ◽

Kamlesh Patel ◽

Haydn Munford ◽

Tabeer Alam ◽

...

Keyword(s):

Shear Stress ◽

High Throughput ◽

Fluid Shear Stress ◽

Tracking System ◽

Frequency Measurement ◽

Camera Motion ◽

Fluid Shear ◽

Mean Frequency

Fluid shear stress is a key modulator of cellular physiology in vitro and in vivo, but its effects are under-investigated due to requirements for complicated induction methods. Herein we report the validation of ShearFAST; a smartphone application that measures the rocking profile on a standard laboratory cell rocker and calculates the resulting shear stress arising in tissue culture plates. The accuracy with which this novel approach measured rocking profiles was validated against a graphical analysis, and also against measures reported by an 8-camera motion tracking system. ShearFASTs angle assessments correlated well with both analyses (r ≥0.99, p ≤0.001) with no significant differences in pitch detected across the range of rocking angles tested. Rocking frequency assessment by ShearFAST also correlated well when compared to the two independent validatory techniques (r ≥0.99, p ≤0.0001), with excellent reproducibility between ShearFAST and video analysis (mean frequency measurement difference of 0.006 ± 0.005Hz) and motion capture analysis (mean frequency measurement difference of 0.008 ± 0.012Hz). These data make the ShearFAST assisted cell rocker model make it an attractive approach for economical, high throughput fluid shear stress experiments. Proof of concept data presented reveals a protective effect of low-level shear stress on renal proximal tubule cells submitted to simulations of pretransplant storage.

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CDK10 Regulates Gastric Cancer Metastasis By Inhibiting lncRNA-C5ORF42-5 Via Phosphorylation Level of AKT/ERK

10.21203/rs.3.rs-610700/v1 ◽

2021 ◽

Author(s):

Zi-Jian Deng ◽

Dong-Wen Chen ◽

Xi-Jie Chen ◽

Jia-Ming Fang ◽

Liang Xv ◽

...

Keyword(s):

Gastric Cancer ◽

Cancer Cells ◽

Cell Line ◽

High Throughput ◽

Cancer Metastasis ◽

High Throughput Sequencing ◽

Gastric Cancer Cells ◽

Related Proteins

Abstract Background: Gastric cancer is the fourth most common malignant disease. Both CDK10 and long noncoding RNAs (lncRNAs) have been found to exert biological functions in multiple cancers. However, it is still unclear whether CDK10 represses tumor progression in gastric cancer by reducing potential targeting lncRNAs.Methods: The functions of CDK10 and lncRNA-C5ORF42-5 in proliferation, invasion and migration were assessed by MTS assays, colony formation assays, cell cycle and apoptosis assays, Transwell assays, wound healing assays and animal experiments. We used high-throughput sequencing to confirm the existence of lncRNA-C5ORF42-5 and quantitative real-time PCR was used to evaluate lncRNA expression. Then, with RNA-seq sequencing as well as GO function and KEGG enrichment analysis, we identified the signaling pathways in which lncRNA-C5ORF42-5 was involved in gastric cancer. Finally, western blotting was used to identify the genes regulated by lncRNA-C5ORF42-5.Results: Our results showed that CDK10 is expressed at relatively low levels in gastric cancer cell lines and inhibits the progression of gastric cancer cells both in vitro and in vivo. Next, based on high-throughput sequencing, we identified a novel lncRNA, lncRNA-C5ORF42-5, in the stable CDK10-overexpressing cell line compared with the CDK-knockdown cell line and their controls. Additionally, we confirmed that lncRNA-C5ORF42-5 acts as an oncogene to promote metastasis in gastric cancer in vitro and in vivo. We then ascertained that lncRNA-C5ORF42-5 is a major contributor to the function of CDK10 in gastric cancer metastasis by upregulating lncRNA-C5ORF42-5 to reverse the effects of CDK10 overexpression. Finally, we explored the mechanism by which lncRNA-C5ORF42-5 overexpression affects gastric cancer cells to elucidate whether lncRNA-C5ORF42-5 may increase the activity of the SMAD pathway of BMP signaling and promote the expression of EMT-related proteins, such as E-cadherin. Additionally, overexpression of lncRNA-C5ORF42-5 affected the phosphorylation levels of AKT and ERK.Conclusion: Our findings suggest that CDK10 overexpression represses gastric cancer tumor progression by reducing lncRNA-C5ORF42-5 and hindering activation of the related proteins in metastatic signaling pathways, which provides new insight into developing effective therapeutic strategies in the treatment of metastatic gastric cancer.

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DIPG-07. HIGH THROUGHPUT DRUG SCREENING IDENTIFIES POTENTIAL NEW THERAPIES FOR DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPGs)

Neuro-Oncology ◽

10.1093/neuonc/noaa222.058 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii288-iii288

Author(s):

Dannielle Upton ◽

Santosh Valvi ◽

Jie Liu ◽

Nicole Yeung ◽

Sandra George ◽

...

Keyword(s):

High Throughput ◽

Apoptotic Cell ◽

Safety Data ◽

Biologically Active ◽

Orthotopic Model ◽

High Throughput Drug Screening ◽

Diffuse Intrinsic Pontine Gliomas ◽

Almost All

Abstract DIPGs are the most devastating of all brain tumors. There are no effective treatments, hence almost all children will die of their tumor within 12 months. There is an urgent need for novel effective therapies for this aggressive tumor. We performed a high-throughput drug screen with over 3,500 biologically active, clinically approved compounds against a panel of neurosphere-forming DIPG cells. We identified 7 compounds- auranofin, fenretinide, ivermectin, lanatoside, parthenolide, SAHA and mefloquine- that were confirmed to have potent anti-tumor activity against a panel of DIPG-neurospheres, with minimal effect on normal cells. Using cytotoxicity and clonogenic assays, we found that these drugs were able to inhibit DIPG-neurosphere proliferation and colony formation in-vitro. To determine whether the in-vitro efficacy could be replicated in-vivo, we tested the activity of each of these compounds in an orthotopic DIPG model. Of the agents tested, fenretinide and SAHA were the most active anti-tumor agents, significantly enhancing the survival of tumor bearing animals. Mechanistic studies showed fenretinide enhancing apoptotic cell death of DIPG cells via inhibition of PDGFRa transcription and downregulation of the PI3K/AKT/MTOR pathway. We therefore examined the therapeutic efficacy of fenretinide using a second orthotopic model with PDGFRa amplification. We used two different Fenretinide formulations (LYM-X-Sorb and NanoMicelle) which were found to enhance survival. Fenretinide is clinically available with safety data in children. Validation of the activity of Fenretinide in PDGFRa-amplified or overexpressed DIPGs will lead to the development of a clinical trial, allowing the advancement of fenretinide as potentially the first active therapy for DIPG.

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Brain Delivery of Thyrotropin-Releasing Hormone via a Novel Prodrug Approach

Pharmaceutics ◽

10.3390/pharmaceutics11070349 ◽

2019 ◽

Vol 11 (7) ◽

pp. 349 ◽

Cited By ~ 1

Author(s):

Katalin Prokai-Tatrai ◽

Daniel L. De La Cruz ◽

Vien Nguyen ◽

Benjamin P. Ross ◽

Istvan Toth ◽

...

Keyword(s):

Brain Homogenate ◽

Thyrotropin Releasing Hormone ◽

Metabolic Stability ◽

Antidepressant Effect ◽

Brain Delivery ◽

Small Peptides ◽

Releasing Hormone ◽

Membrane Affinity

Using thyrotropin-releasing hormone (TRH) as a model, we explored whether synergistic combination of lipoamino acid(s) and a linker cleaved by prolyl oligopeptidase (POP) can be used as a promoiety for prodrug design for the preferential brain delivery of the peptide. A representative prodrug based on this design principle was synthesized, and its membrane affinity and in vitro metabolic stability, with or without the presence of a POP inhibitor, were studied. The in vivo formation of TRH from the prodrug construct was probed by utilizing the antidepressant effect of the peptide, as well as its ability to increase acetylcholine (ACh) synthesis and release. We found that the prototype prodrug showed excellent membrane affinity and greatly increased metabolic stability in mouse blood and brain homogenate compared to the parent peptide, yet a POP inhibitor completely prevented prodrug metabolism in brain homogenate. In vivo, administration of the prodrug triggered antidepressant-like effect, and microdialysis sampling showed greatly increased ACh release that was also antagonized upon a POP inhibitor treatment. Altogether, the obtained promising exploratory data warrant further investigations on the utility of the prodrug approach introduced here for brain-enhanced delivery of small peptides with neurotherapeutic potential.

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Translation of In Vitro Activity to In Vivo Activity: Lessons from the Triazolopyrimidine Sulfonanilides

Weed Science ◽

10.1017/s0043174500094649 ◽

1996 ◽

Vol 44 (3) ◽

pp. 757-762 ◽

Cited By ~ 1

Author(s):

B. Clifford Gerwick ◽

Csaba T. Cseke ◽

Gerry Deboer ◽

William A. Kleschick ◽

Paul R. Schmitzer

Keyword(s):

Acetolactate Synthase ◽

Metabolic Stability ◽

Plant Structure ◽

Weed Species ◽

Whole Plant ◽

Stability Data ◽

Activity Models ◽

Two Parameter

Eight triazolopyrimidine sulfonanilides were tested for metabolic stability in a number of crop and weed species. These data, along with in vitro determinations of activity (I50) against acetolactate synthase, successfully described the in vivo activity of these compounds in a two-parameter model. Whole plant activity increased with increasing compound stability and decreasing I50 (r2 =.78, N = 36). The difficulty in obtaining metabolic stability data during a structure optimization program prompted a study with substituent parameters in models of in vivo activity. Models describing whole plant activity in jimsonweed were developed using a series of 5-methyl triazolopyrimidine sulfonanilides that differed only in ortho and meta substituents on the aniline ring. The I50 term and clogP were most important to jimsonweed activity. Hence, in vitro activity (I50) may be a useful component of whole plant structure activity models to aid in identification of barriers to in vivo performance.

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HER2 Kinase-Targeted Breast Cancer Therapy: Design, Synthesis, and In Vitro and In Vivo Evaluation of Novel Lapatinib Congeners as Selective and Potent HER2 Inhibitors with Favorable Metabolic Stability

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.0c01647 ◽

2020 ◽

Vol 63 (24) ◽

pp. 15906-15945

Author(s):

Tamer A. Elwaie ◽

Safinaz E. Abbas ◽

Enayat I. Aly ◽

Riham F. George ◽

Hamdy Ali ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Therapy ◽

Metabolic Stability ◽

Breast Cancer Therapy ◽

In Vivo Evaluation ◽

Design Synthesis ◽

Therapy Design

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T cell neoepitope discovery in colorectal cancer by high throughput profiling of somatic mutations in expressed genes

Gut ◽

10.1136/gutjnl-2015-309453 ◽

2015 ◽

Vol 66 (3) ◽

pp. 454-463 ◽

Cited By ~ 24

Author(s):

Daniele Mennonna ◽

Cristina Maccalli ◽

Michele C Romano ◽

Claudio Garavaglia ◽

Filippo Capocefalo ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

High Throughput ◽

High Throughput Sequencing ◽

Cell Epitope ◽

Patient Specific ◽

Cancer Genes ◽

Healthy Donors

ObjectivePatient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies.DesignWe undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions.ResultsSeveral unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+and CD4+T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8+T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo.ConclusionsThese results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.

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