Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

Tel Aviv University

Polymer conjugation is an efficient approach to improve the delivery of drugs and biological agents, both by protecting the body from the drug (by improving biodistribution and reducing toxicity) and by protecting the drug from the body (by preventing degradation and enhancing cellular uptake). This review discusses the journey that polymer therapeutics make through the body, following the ADME (absorption, distribution, metabolism, excretion) concept. The biological factors and delivery system parameters that influence each stage of the process will be described, with examples illustrating the different solutions to the challenges of drug delivery systems in vivo.

Administration, distribution, metabolism and elimination of polymer therapeutics.

Although dormant tumors are highly prevalent within the human population, the underlying mechanisms are still mostly unknown. We have previously identified the consensus gene expression pattern of dormant tumors. Here, we show that this gene expression signature could be used for the isolation and identification of clones which generate dormant tumors. We established single cell-derived clones from the aggressive tumor-generating U-87 MG human glioblastoma cell line. Based only on the expression pattern of genes which were previously shown to be associated with tumor dormancy, we identified clones which generate dormant tumors. We show that very high expression levels of thrombospondin and high expression levels of angiomotin and insulin-like growth factor binding protein 5 (IGFBP5), together with low levels of endothelial specific marker (ESM) 1 and epithelial growth factor receptor (EGFR) characterize the clone which generates dormant U-87 MG derived glioblastomas. These tumors remained indolent both in subcutaneous and orthotopic intracranial sites, in spite of a high prevalence of proliferating cells. We further show that tumor cells which form U-87 MG derived dormant tumors have an impaired angiogenesis potential both in vitro and in vivo and have a slower invasion capacity. This work demonstrates that fast-growing tumors contain tumor cells that when isolated will form dormant tumors and serves as a proof-of-concept for the use of transcriptome profiles in the identification of such cells. Isolating the tumor cells that form dormant tumors will facilitate understanding of the underlying mechanisms of dormant micro-metastases, late recurrence, and changes in rate of tumor progression.

Prospective identification of glioblastoma cells generating dormant tumors.

This protocol describes the synthesis of modular turn-ON QCy7-based probes for the detection of biologically relevant analytes, such as hydrogen peroxide, ubiquitous sulfhydryl and β-galactosidase. The probes presented herein are prepared through a simple procedure that involves the preliminary alkylation of 4-hydroxy-isophthalaldehyde with a relevant analyte-responsive protecting group, followed by condensation of the resulting product with 2 equivalents of sulfo-indolium moieties. Evaluation of the turn-ON near-IR fluorescence response to their relevant analytes for the three different QCy7 probes is also reported. The preparation of a QCy7 diagnostic probe requires 1-2 d. Probes for other analytes can be prepared according to this modular procedure by incorporating a specific analyte-responsive group as a triggering substrate.

Synthesis and use of QCy7-derived modular probes for the detection and imaging of biologically relevant analytes.

Polymeric nanocarriers conjugated with low molecular weight drugs are designed in order to improve their efficacy and toxicity profile. This approach is particularly beneficial for anticancer drugs, where the polymer-drug conjugates selectively accumulate at the tumor site, due to the enhanced permeability and retention (EPR) effect. The conjugated drug is typically inactive, and upon its pH- or enzymatically-triggered release from the carrier, it regains its therapeutic activity. These settings lack information regarding drug-release time, kinetics and location. Thereby, real-time non-invasive intravital monitoring of drug release is required for theranostics (therapy and diagnostics). We present here the design, synthesis and characterization of a theranostic nanomedicine, based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, owing its fluorescence-based monitoring of site-specific drug release to a self-quenched near-infrared fluorescence (NIRF) probe. We designed two HPMA copolymer-based systems that complement to a theranostic nanomedicine. The diagnostic system consists of self-quenched Cy5 (SQ-Cy5) as a reporter probe and the therapeutic system is based on the anticancer agent paclitaxel (PTX). HPMA copolymer-PTX/SQ-Cy5 systems enable site-specific release upon enzymatic degradation in cathepsin B-overexpressing breast cancer cells. The release of the drug occurs concomitantly with the activation of the fluorophore to its Turn-ON state. HPMA copolymer-SQ-Cy5 exhibits preferable body distribution and drug release compared with the free drug and probe when administered to cathepsin B-overexpressing 4T1 murine mammary adenocarcinoma-bearing mice. This approach of co-delivery of two complementary systems serves as a proof-of-concept for real-time deep tissue intravital orthotopic monitoring and may have the potential use in clinical utility as a theranostic nanomedicine.

Polymeric nanotheranostics for real-time non-invasive optical imaging of breast cancer progression and drug release.

Recent years have seen tremendous progress in the design and study of molecular imaging geared towards biological and biomedical applications. The expression or activity of specific enzymes including proteases can be monitored by cutting edge molecular imaging techniques. Cathepsin B plays key roles in tumor progression via controlled degradation of extracellular matrix. Consequently, this protease has been attracting significant attention in cancer research, and many imaging probes targeting its activity have been developed. Here, we describe the design, synthesis and evaluation of two novel near infrared (NIR) fluorescent probes for detection of cathepsin B activity with different turn-ON mechanisms. One probe is based on an ICT activation mechanism of a donor-two-acceptor π-electron dye system, while the other is based on the FRET mechanism obtained by a fluorescent dye and a quencher. The two probes exhibit significant fluorescent turn-ON response upon cleavage by cathepsin B. The NIR fluorescence of the ICT probe in its OFF state was significantly lower than that of the FRET-based probe. This effect results in a higher signal-to-noise ratio and consequently increased sensitivity and better image contrast.

Synthesis and evaluation of new NIR-fluorescent probes for cathepsin B: ICT versus FRET as a turn-ON mode-of-action.

Protein phosphatase magnesium dependent 1A (PPM1A) has been implicated in fibrosis and skin wounding. We generated PPM1A knockout mice to study the role of PPM1A in the wound healing-inflammation-angiogenesis cross talk. The role of PPM1A in these processes was studied using the ocular alkali burn model system. In the injured cornea the absence of PPM1A led to enhanced inflammatory response, stromal keratocyte transactivation, fibrosis, increased p38 mitogen-activated protein kinase phosphorylation, elevated expression of transforming growth factor-β-related genes (including Acta2, TGF-β, Col1, MMP9, and VEGF) and subsequently to neovascularization. Augmented angiogenesis in the absence of PPM1A is a general process occurring in vivo in PPM1A knockout mice upon subcutaneous Matrigel injection and ex vivo in aortic ring Matrigel cultures. Using primary keratocyte cultures and various experimental approaches, we found that phospho-p38 is a favored PPM1A substrate and that by its dephosphorylation PPM1A participates in the regulation of the transforming growth factor-β signaling cascade, the hallmark of inflammation and the angiogenic process. On the whole, the studies presented here position PPM1A as a new player in the wound healing-inflammation-angiogenesis axis in mouse, reveal its crucial role in homeostasis on injury, and highlight its potential as a therapeutic mediator in pathologic conditions, such as inflammation and angiogenesis disorders, including cancer.

Shiran Ferber

Department of Physiology and Pharmacology,
Sackler School of Medicine,
Department of Physiology and Pharmacology, Sackler School of Medicine

Tel Aviv University

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

Shiran Ferber

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physiology and Pharmacology,Sackler School of Medicine,Department of Physiology and Pharmacology, Sackler School of Medicine

Tel Aviv University

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

Department of Physiology and Pharmacology,
Sackler School of Medicine,
Department of Physiology and Pharmacology, Sackler School of Medicine