scholarly journals Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies

Pharmaceutics ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1390
Author(s):  
Bruce Howng ◽  
Michael B. Winter ◽  
Carol LePage ◽  
Irina Popova ◽  
Michael Krimm ◽  
...  
Keyword(s):  
Protease Activity ◽  
Ex Vivo ◽  
Human Tumor ◽  
Cancer Therapeutics ◽  
Biological Tissues ◽  
Clinical Samples ◽  
Reliable Technique ◽  
Physiological Processes ◽  
Wide Range ◽  
Therapeutic Development

Proteases are involved in the control of numerous physiological processes, and their dysregulation has been identified in a wide range of pathologies, including cancer. Protease activity is normally tightly regulated post-translationally and therefore cannot be accurately estimated based on mRNA or protein expression alone. While several types of zymography approaches to estimate protease activity exist, there remains a need for a robust and reliable technique to measure protease activity in biological tissues. We present a novel quantitative ex vivo zymography (QZ) technology based on Probody® therapeutics (Pb-Tx), a novel class of protease-activated cancer therapeutics that contain a substrate linker cleavable by tumor-associated proteases. This approach enables the measurement and comparison of protease activity in biological tissues via the detection of Pb-Tx activation. By exploiting substrate specificity and selectivity, cataloguing and differentiating protease activities is possible, with further refinement achieved using protease-specific inhibitors. Using the QZ assay and human tumor xenografts, patient tumor tissues, and patient plasma, we characterized protease activity in preclinical and clinical samples. The QZ assay offers the potential to increase our understanding of protease activity in tissues and inform diagnostic and therapeutic development for diseases, such as cancer, that are characterized by dysregulated proteolysis.

scholarly journals Antitumoral Effect of Laurinterol on 3D Culture of Breast Cancer Explants

Marine Drugs ◽  
2019 ◽  
Vol 17 (4) ◽  
pp. 201 ◽  
Author(s):  
Sara García-Davis ◽  
Ezequiel Viveros-Valdez ◽  
Ana Díaz-Marrero ◽  
José Fernández ◽  
Daniel Valencia-Mercado ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Crude Extract ◽  
Human Breast Cancer ◽  
Ex Vivo ◽  
3D Culture ◽  
Human Tumor ◽  
Individual Response ◽  
Human Breast ◽  
Antitumoral Effect ◽  
Wide Range

Macroalgae represent an important source of bioactive compounds with a wide range of biotechnological applications. Overall, the discovery of effective cytotoxic compounds with pharmaceutical potential is a significant challenge, mostly because they are scarce in nature or their total synthesis is not efficient, while the bioprospecting models currently used do not predict clinical responses. Given this context, we used three-dimensional (3D) cultures of human breast cancer explants to evaluate the antitumoral effect of laurinterol, the major compound of an ethanolic extract of Laurencia johnstonii. To this end, we evaluated the metabolic and histopathological effects of the crude extract of L. johnstonii and laurinterol on Vero and MCF-7 cells, in addition to breast cancer explants. We observed a dose-dependent inhibition of the metabolic activity, as well as morphologic and nuclear changes characteristic of apoptosis. On the other hand, a reduced metabolic viability and marked necrosis areas were observed in breast cancer explants incubated with the crude extract, while explants treated with laurinterol exhibited a heterogeneous response which was associated with the individual response of each human tumor sample. This study supports the cytotoxic and antitumoral effects of laurinterol in in vitro cell cultures and in ex vivo organotypic cultures of human breast cancer explants.

Biomarker-driven indication selection in JTX-2011 ICONIC clinical trial.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 11602-11602
Author(s):  
Heather Anne Hirsch ◽  
Jason Reeves ◽  
Tong Zi ◽  
Amit Deshpande ◽  
Guang Yang ◽  
...  
Keyword(s):  
Solid Tumors ◽  
In Silico ◽  
Immune Cell ◽  
Ex Vivo ◽  
Single Agent ◽  
Predictive Biomarkers ◽  
Clinical Samples ◽  
Integrated Analysis ◽  
T Effector Cells ◽  
Wide Range

11602 Background: ICOS (Inducible T cell CO-Stimulator) is a co-stimulatory molecule expressed primarily on T lymphocytes. Clinical and preclinical data suggest that ICOS mediates anti-CTLA-4 driven anti-tumor responses. JTX-2011 is an ICOS agonist antibody in clinical development in advanced solid tumors (ICONIC trial). JTX-2011 is designed to generate an anti-tumor immune response via stimulation of T effector cells and preferential reduction of intra-tumoral T regulatory cells. Single agent preclinical efficacy correlates with the percentage of ICOS-expressing T cells within the tumor. We report indication selection and patient enrichment strategy for ICONIC using in silico and IHC analysis and assessment of potential predictive biomarkers for JTX-2011 using ex vivo tumor histoculture. Methods: Integrated analysis was performed from the TCGA for ICOS expression in histologic and molecularly defined tumors and immune cell signatures. ICOS expression was analyzed by IHC in a subset of indications based on in silico analysis. ICOS expression on intra-tumoral Tregs and PD-L1 were analyzed in a cohort of 126 head and neck squamous cell carcinomas (HNSCC). Ex vivo histoculture assays of human HNSCC was treated with JTX-2011 and assessed for IFNg gene signature induction. Results: ICOS mRNA expression was analyzed in ~10,000 solid tumors samples across ~30 indications. ICOS expression in key indications was confirmed using IHC. Based on frequency of high ICOS expression, non-small cell lung cancer, HNSCC, triple negative breast carcinoma, gastric cancer, and melanoma were selected as indications for ICONIC. Results were confirmed in clinical samples using multiplex immunofluorescence and ICOS IHC. A wide range of ICOS expression was observed suggesting that identification of an ICOS “high” group may enrich for patients likely to benefit from ICOS agonist therapy. In ex vivo histoculture assays of human HNSCC tumors treated with JTX-2011, ICOS IHC and ICOS RNA gene signatures correlated to response endpoints. Comparison of ICOS and PDL1 expression identified subsets of tumors in multiple indications with high ICOS but low PDL1 expression. Conclusions: These data support prioritization of specific tumor types the ICONIC trial.

scholarly journals Single Cell Analysis of Regions of Interest (SCARI) using a novel photoswitchable tag

2020 ◽  
Author(s):  
Anne M. van der Leun ◽  
Mirjam E. Hoekstra ◽  
Luuk Reinalda ◽  
Colinda L.G.J. Scheele ◽  
Mireille Toebes ◽  
...  
Keyword(s):  
Single Cell ◽  
Spatial Information ◽  
Ex Vivo ◽  
Environmental Influence ◽  
Human Tumor ◽  
Regions Of Interest ◽  
Live Cells ◽  
Clinical Samples ◽  
Differentiation Potential ◽  
Depth Analysis

AbstractThe functional activity and differentiation potential of cells is determined by their interaction with surrounding cells. Approaches that allow the unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a trade-off between spatial resolution and cell profiling depth. Here, we developed a photoswitch-based technology that allows the isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran)-cage coupled to nanobodies allowed photoswitching of cells in areas of interest with low-intensity violet light and without detectable phototoxicity. Single cell RNA sequencing of spatially defined CD8+ T cells was used to exemplify the feasibility of identifying location-dependent cell states at the single cell level. Finally, we demonstrate the efficient labeling and photoswitching of cells in live primary human tumor tissue. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples.

scholarly journals Ex-Vivo Exposure on Biological Tissues in the 2-μm Spectral Range with an All-Fiber Continuous-Wave Holmium Laser

Photonics ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 20
Author(s):  
Mariya S. Kopyeva ◽  
Serafima A. Filatova ◽  
Vladimir A. Kamynin ◽  
Anton I. Trikshev ◽  
Elizaveta I. Kozlikina ◽  
...  
Keyword(s):  
Solid State ◽  
Soft Tissues ◽  
Continuous Wave ◽  
Ex Vivo ◽  
Laser System ◽  
Biological Tissues ◽  
Holmium Laser ◽  
Experimental Point ◽  
Ablation Efficiency ◽  
Wide Range

We present the results on the interaction of an all-fiber Holmium-doped laser CW radiation at a wavelength of 2100 nm with soft tissues and compare it with the other results obtained by the most used solid-state laser systems. Ex-vivo single spot experiments were carried out on the porcine longissimus muscles by varying the laser impact parameters in a wide range (average output power 0.3, 0.5 and 1.1 W; exposure time 5, 30 and 60 s). Evaluation of the laser radiation exposure was carried out by the size of coagulation and ablation zones on the morphological study. Exposure to a power of 0.3 W (1.5–18 J of applied energy) caused only reversible changes in the tissues. The highest applied energy of 66 J for 1.1 W and a 60-s exposure resulted in a maximum ablation depth of approximately 1.2 mm, with an ablation efficiency of 35%. We have shown that it is not necessary to use high powers of CW radiation, such as 5–10 W in the solid-state systems to provide the destructive effects. Similar results can be achieved at lower powers using the simple all-fiber Holmium laser based on the standard single-mode fiber, which could provide higher power densities and be more convenient to manufacture and use. The obtained results may be valuable as an additional experimental point in the field of existing results, which in the future will allow one to create a simple optimal laser system for medical purposes.

The endocannabinoid system

2020 ◽  
Vol 64 (3) ◽  
pp. 485-499
Author(s):  
Aruna Kilaru ◽  
Kent D. Chapman
Keyword(s):  
Signaling Pathways ◽  
Membrane Lipids ◽  
Endocannabinoid System ◽  
Physiological Processes ◽  
Therapeutic Value ◽  
Wide Range ◽  
Therapeutic Development ◽  
The Central Nervous System ◽  
Psychotropic Effects ◽  
Signaling Process

Abstract Thirty years ago, the discovery of a cannabinoid (CB) receptor that interacts with the psychoactive compound in Cannabis led to the identification of anandamide, an endogenous receptor ligand or endocannabinoid. Research on endocannabinoids has since exploded, and additional receptors along with their lipid mediators and signaling pathways continue to be revealed. Specifically, in humans, the release of endocannabinoids from membrane lipids occurs on demand and the signaling process is rapidly attenuated by the breakdown of the ligand suggesting a tight regulation of the endocannabinoid system (ECS). Additionally, the varying distribution of CB receptors between the central nervous system and other tissues allows for the ECS to participate in a wide range of cognitive and physiological processes. Select plant-derived ‘phyto’cannabinoids such as Δ-9-tetrahydrocannabinol (Δ9-THC) bind to the CB receptors and trigger the ECS, and in the case of Δ9-THC, while it has therapeutic value, can also produce detrimental effects. Current research is aimed at the identification of additional phytocannabinoids with minimal psychotropic effects with potential for therapeutic development. Although decades of research on the ECS and its components have expanded our understanding of the mechanisms and implications of endocannabinoid signaling in mammals, it continues to evolve. Here, we provide a brief overview of the ECS and its overlap with other related lipid-mediated signaling pathways.

scholarly journals Association of Blastocystis ST6 with higher protease activity among symptomatic subjects

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Seyed Ahmad Karamati ◽  
Hamed Mirjalali ◽  
Maryam Niyyati ◽  
Abbas Yadegar ◽  
Hamid Asadzadeh Aghdaei ◽  
...  
Keyword(s):  
Cell Line ◽  
Protease Activity ◽  
Clinical Symptoms ◽  
Protozoan Parasite ◽  
Inflammatory Biomarkers ◽  
Clinical Samples ◽  
Intestinal Protozoan ◽  
Related Factors ◽  
Wide Range ◽  
Ht 29

Abstract Background Blastocystis sp. is an anaerobic intestinal protozoan parasite of humans and a wide range of animals worldwide. In the current study the correlation between the cysteine protease activity of clinical samples of Blastocystis sp. ST1–3 and 6 with the levels of pro-inflammatory cytokines was evaluated. Methods Stool samples were collected from subjects with or without clinical symptoms. All samples were cultivated in DMEM medium. The bacteria were eliminated or reduced in Blastocystis sp. positive samples subtypes 1–3 and 6 by a variety of antibiotics and consecutive sub-cultures. To prepare parasite lysate, 1 × 105Blastocystis sp. from each isolate were harvested and lysed using freeze-thaw. Protease activity of each isolate was measured and the gene expression of pro-inflammatory biomarkers in HT-29 cell line sensed by isolates was investigated using quantitative Real-time PCR. Results Protease activity assay showed inter- and intra-subtype variations among subtypes regarding the presence of symptoms, while the protease activity of symptomatic isolates was higher than asymptomatic isolates. The highest and lowest levels of protease activity were seen in ST6 and ST2, respectively. However, patterns of the expression of pro-inflammatory biomarkers in HT-29 cell line was different regarding the presence of symptoms and time points. There was no significant correlation between protease activity of different subtypes with the expression levels of pro-inflammatory biomarkers. Conclusions Our study indicated a higher protease activity among isolates from symptomatic compared to asymptomatic subjects, suggesting functional role for proteases in clinical symptoms due to Blastocystis sp. The lack of correlation between the levels of expression of pro-inflammatory biomarkers with subtypes regarding the presence of clinical symptoms proposes the importance of host-related factors in presentation of clinical symptoms.

scholarly journals Single Cell Analysis of Regions of Interest (SCARI) using a Novel Photoswitchable Tag

2020 ◽  
Author(s):  
Anne van der Leun ◽  
Mirjam Hoekstra ◽  
Luuk Reinalda ◽  
Colinda Scheele ◽  
Mireille Toebes ◽  
...  
Keyword(s):  
Single Cell ◽  
Spatial Information ◽  
Ex Vivo ◽  
Environmental Influence ◽  
Human Tumor ◽  
Regions Of Interest ◽  
Live Cells ◽  
Clinical Samples ◽  
Differentiation Potential ◽  
Depth Analysis

Abstract The functional activity and differentiation potential of cells is determined by their interaction with surrounding cells. Approaches that allow the unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a trade-off between spatial resolution and cell profiling depth. Here, we developed a photoswitch-based technology that allows the isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran)-cage coupled to nanobodies allowed photoswitching of cells in areas of interest with low-intensity violet light and without detectable phototoxicity. Single cell RNA sequencing of spatially defined CD8+ T cells was used to exemplify the feasibility of identifying location-dependent cell states at the single cell level. Finally, we demonstrate the efficient labeling and photoswitching of cells in live primary human tumor tissue. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples.

scholarly journals Custom Multiphoton/Raman Microscopy Setup for Imaging and Characterization of Biological Samples

2019 ◽  
Vol 2 (2) ◽  
pp. 51 ◽  
Author(s):  
Marco Marchetti ◽  
Enrico Baria ◽  
Riccardo Cicchi ◽  
Francesco Saverio Pavone
Keyword(s):  
Laser Scanning ◽  
Ex Vivo ◽  
Second Harmonic ◽  
Biological Tissues ◽  
Molecular Organization ◽  
Chemical Information ◽  
Specific Information ◽  
Label Free ◽  
Wide Range ◽  
Experimental Apparatus

Modern optics offers several label-free microscopic and spectroscopic solutions which are useful for both imaging and pathological assessments of biological tissues. The possibility to obtain similar morphological and biochemical information with fast and label-free techniques is highly desirable, but no single optical modality is capable of obtaining all of the information provided by histological and immunohistochemical analyses. Integrated multimodal imaging offers the possibility of integrating morphological with functional-chemical information in a label-free modality, complementing the simple observation with multiple specific contrast mechanisms. Here, we developed a custom laser-scanning microscopic platform that combines confocal Raman spectroscopy with multimodal non-linear imaging, including Coherent Anti-Stokes Raman Scattering, Second-Harmonic Generation, Two-Photon Excited Fluorescence, and Fluorescence Lifetime Imaging Microscopy. The experimental apparatus is capable of high-resolution morphological imaging of the specimen, while also providing specific information about molecular organization, functional behavior, and molecular fingerprint. The system was successfully tested in the analysis of ex vivo tissues affected by urothelial carcinoma and by atherosclerosis, allowing us to multimodally characterize of the investigated specimen. Our results show a proof-of-principle demonstrating the potential of the presented multimodal approach, which could serve in a wide range of biological and biomedical applications.

scholarly journals Simulating the human tumor microenvironment in colorectal cancer organoids in vitro and in vivo

2019 ◽  
Author(s):  
Mahesh Devarasetty ◽  
Anthony Dominijanni ◽  
Samuel Herberg ◽  
Ethan Shelkey ◽  
Aleksander Skardal ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Tumor Microenvironment ◽  
Cancer Progression ◽  
Ex Vivo ◽  
Human Tumor ◽  
Cancer Therapeutics ◽  
Low Grade ◽  
Ecm Architecture

AbstractThe tumor microenvironment (TME) plays a significant role in cancer growth and metastasis. Bioengineered models of the TME will advance our understanding of cancer progression and facilitate identification of novel anti-cancer therapeutics that target TME components such as extracellular matrix (ECM) and stromal cells. However, most current in vitro models fail to recapitulate the extensive features of the human tumor stroma, especially ECM architecture, and are not exposed to intact body physiology. On the other hand, in vivo animal models do not accurately capture human tumor architecture. Using the features of biopsied colorectal cancer (CRC) tissue as a guide, we address these deficiencies by creating human organoids containing a colonic stromal ECM layer and CRC spheroids. Organoids were studied in vitro and upon implantation in mice for 28 days. We show that the stromal ECM micro-architecture, generated in vitro, was maintained in vivo for at least 28 days. Furthermore, comparisons with biopsied CRC tumors revealed that organoids with orderly structured TMEs induce an epithelial phenotype in CRC cells, similar to low-grade tumors, compared to a mesenchymal phenotype observed in disordered TMEs, similar to high-grade tumors. Altogether, these results are the first demonstration of replicating the human tumor ECM architecture in biofabricated tumor organoids under ex vivo and in vivo conditions.

scholarly journals Transfer of Plasmid DNA to Clinical Coagulase-Negative Staphylococcal Pathogens by Using a Unique Bacteriophage

2015 ◽  
Vol 81 (7) ◽  
pp. 2481-2488 ◽  
Author(s):  
Volker Winstel ◽  
Petra Kühner ◽  
Bernhard Krismer ◽  
Andreas Peschel ◽  
Holger Rohde
Keyword(s):  
Plasmid Dna ◽  
Genetic Manipulation ◽  
Current Knowledge ◽  
Virulence Determinants ◽  
Coagulase Negative Staphylococci ◽  
Reliable Technique ◽  
Content Type ◽  
Research Activities ◽  
Wide Range ◽  
Genetic Barriers

ABSTRACTGenetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a uniqueStaphylococcus aureusstrain via a specificS. aureusbacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinicalStaphylococcus epidermidisisolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

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