Simultaneous selection of nanobodies for accessible epitopes on immune cells in the tumor microenvironment.

In the rapidly advancing field of synthetic biology, there exists a critical need for technology to discover targeting moieties for therapeutic biologics. Here we present INSPIRE-seq, an approach that utilizes a nanobody library and next-generation sequencing to identify nanobodies selected for complex environments. INSPIRE-seq enables the parallel enrichment of immune cell-binding nanobodies that penetrate the tumor microenvironment.

Restoration of the Lost Human Beta Defensin (hBD-1) in Cancer as a Strategy to Improve the Efficacy of Chemotherapy.

Both innate and adaptive immunity are the important components of the human defense system against various diseases including cancer. Human Beta Defensin (hBD-1) is one such immunomodulatory peptide which is lost at high frequencies in malignant cancers, while high levels of expression are maintained in benign regions making it a potential biomarker for the onset and metastasis of the disease. Loss of putative function of hBD-1 as a tumor suppressor gene combined with the defects in apoptosis pathways (CD95, ASK1) make tumor cells insensitive to chemotherapy and render it ineffective.

Ultrasound-guided delivery of thymidine kinase-nitroreductase dual therapeutic genes by PEGylated-PLGA/PIE nanoparticles for enhanced triple negative breast cancer therapy.

Aim: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype. Since no targeted therapy is available, gene-directed enzyme prodrug therapy (GDEPT) could be an attractive strategy for treating TNBC.

Materials & Methods: Polyethylene glycol (PEG)ylated-poly(lactic-co-glycolic acid)/polyethyleneimine nanoparticles (PLGA/PEI NPs) were synthesized and complexed with TK-NTR fusion gene.

A protein folding molecular imaging biosensor monitors the effects of drugs that restore mutant p53 structure and its downstream function in glioblastoma cells.

Misfolding mutations in the DNA-binding domain of p53 alter its conformation, affecting the efficiency with which it binds to chromatin to regulate target gene expression and cell cycle checkpoint functions in many cancers, including glioblastoma. Small molecule drugs that recover misfolded p53 structure and function may improve chemotherapy by activating p53-mediated senescence. We constructed and optimized a split luciferase (RLUC) complementation molecular biosensor (NRLUC-p53-CRLUC) to determine small molecule-meditated folding changes in p53 protein.

Targeted nanoparticle delivery of therapeutic antisense microRNAs presensitizes glioblastoma cells to lower effective doses of temozolomide and in a mouse model.

Temozolomide (TMZ) chemotherapy for glioblastoma (GBM) is generally well tolerated at standard doses but it can cause side effects. GBMs overexpress microRNA-21 and microRNA-10b, two known oncomiRs that promote cancer development, progression and resistance to drug treatment. We hypothesized that systemic injection of antisense microRNAs (antagomiR-21 and antagomiR-10b) encapsulated in cRGD-tagged PEG-PLGA nanoparticles would result in high cellular delivery of intact functional antagomiRs, with consequent efficient therapeutic response and increased sensitivity of GBM cells to lower doses of TMZ.

Engineering Intracellularly Retained Gaussia Luciferase Reporters for Improved Biosensing and Molecular Imaging Applications.

Gaussia luciferase (GLUC) is a bioluminescent reporter protein of increasing importance. As a secretory protein, it has increased sensitivity in vitro and in vivo (∼20 000-fold, and ∼1000-fold, respectively) over its competitor, secreted alkaline phosphatase. Unfortunately, this same advantageous secretory nature of GLUC limits its usefulness for many other possible intracellular applications, e.

Gemcitabine and Antisense-microRNA Co-encapsulated PLGA-PEG Polymer Nanoparticles for Hepatocellular Carcinoma Therapy.

Hepatocellular carcinoma (HCC) is highly prevalent, and the third most common cause of cancer-associated deaths worldwide. HCC tumors respond poorly to chemotherapeutic anticancer agents due to inherent and acquired drug resistance, and low drug permeability. Targeted drug delivery systems with significant improvement in therapeutic efficiency are needed for successful HCC therapy.

Molecular Imaging Biosensor Monitors p53 Sumoylation in Cells and Living Mice.

Small molecule mediated stabilization of p53 tumor suppressor protein through sumoylation is a promising new strategy for improving cancer chemotherapy. A molecular tool that monitors p53 sumoylation status and expedites screening for drugs that enhance p53 sumoylation would be beneficial. We report a molecularly engineered reporter fragment complementation biosensor based on optical imaging of Firefly luciferase (FLuc), to quantitatively image p53 sumoylation and desumoylation in cells and living mice.

A transgenic mouse model expressing an ERα folding biosensor reveals the effects of Bisphenol A on estrogen receptor signaling.

Estrogen receptor-α (ERα) plays an important role in normal and abnormal physiology of the human reproductive system by interacting with the endogenous ligand estradiol (E2). However, other ligands, either analogous or dissimilar to E2, also bind to ERα. This may create unintentional activation of ER signaling in reproductive tissues that can lead to cancer development.

Theranostic Imaging of Cancer Gene Therapy.

Gene-directed enzyme prodrug therapy (GDEPT) is a promising therapeutic approach for treating cancers of various phenotypes. This strategy is independent of various other chemotherapeutic drugs used for treating cancers where the drugs are mainly designed to target endogenous cellular mechanisms, which are different in various cancer subtypes. In GDEPT an external enzyme, which is different from the cellular proteins, is expressed to convert the injected prodrug in to a toxic metabolite, that normally kill cancer cells express this protein.

Imaging Histone Methylations in Living Animals.

Histone modifications (methylation, acetylation, phosphorylation, sumoylation, etc.,) are at the heart of cellular regulatory mechanisms, which control expression of genes in an orderly fashion and control the entire cellular regulatory networks. Histone lysine methylation has been identified as one of the several posttranslational histone modifications that plays crucial role in regulating gene expressions in facultative heterochromatic DNA regions while maintaining structural integrity in constitutive heterochromatic DNA regions.

A molecular imaging biosensor detects in vivo protein folding and misfolding.

Unlabelled: Aberrant protein folding represents the molecular basis of many important human diseases. Although the discovery of new anti-misfolding drugs is a major priority in molecular therapeutics, there is currently no generalizable protein folding assay for use in cell-based high throughput screening (HTS) of chemical libraries, or for in vivo imaging. We molecularly engineered a bioluminescence-based biosensor composed of rationally split Firefly luciferase reporter fragments flanking a test protein, and used this in a protein-fragment complementation assay to quantitatively measure folding of the test protein.

Folate Receptor-Targeted Polymeric Micellar Nanocarriers for Delivery of Orlistat as a Repurposed Drug against Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a recalcitrant malignancy with no available targeted therapy. Off-target effects and poor bioavailability of the FDA-approved antiobesity drug orlistat hinder its clinical translation as a repurposed new drug against TNBC. Here, we demonstrate a newly engineered drug formulation for packaging orlistat tailored to TNBC treatment.

Monitoring the Antioxidant Mediated Chemosensitization and ARE-Signaling in Triple Negative Breast Cancer Therapy.

Chemotherapy often fails due to cellular detoxifying mechanisms, including phase-II enzymes. Activation of Nrf2-Keap1 pathway induces phase-II enzymes expression through ARE-signaling and prevents cancer development. Nrf2-overexpression in cancer cells results in chemo- and/or radioresistance.

Nrf2 activity as a potential biomarker for the pan-epigenetic anticancer agent, RRx-001.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulatory transcription factor that plays an important role in the antioxidant response pathway against anticancer drug-induced cytotoxic effects. RRx-001 is a new anticancer agent that generates reactive oxygen and nitrogen species, and leads to epigenetic alterations in cancer cells. Here we report the RRx-001 mediated nuclear translocation of Nrf2 and the activation of expression of its downstream enzymes HO-1 and NQO1 in tumor cells.

Formulation of Anti-miR-21 and 4-Hydroxytamoxifen Co-loaded Biodegradable Polymer Nanoparticles and Their Antiproliferative Effect on Breast Cancer Cells.

Breast cancer is the second leading cause of cancer-related death in women. The majority of breast tumors are estrogen receptor-positive (ER+) and hormone-dependent. Neoadjuvant anti-estrogen therapy has been widely employed to reduce tumor mass prior to surgery.

Polymer nanoparticles mediated codelivery of antimiR-10b and antimiR-21 for achieving triple negative breast cancer therapy.

The current study shows the therapeutic outcome achieved in triple negative breast cancer (TNBC) by simultaneously antagonizing miR-21-induced antiapoptosis and miR-10b-induced metastasis, using antisense-miR-21-PS and antisense-miR-10b-PS delivered by polymer nanoparticles (NPs). We synthesized the antisense-miR-21 and antisense-miR-10b loaded PLGA-b-PEG polymer NPs and evaluated their cellular uptake, serum stability, release profile, and the subsequent synchronous blocking of endogenous miR-21 and miR-10b function in TNBC cells in culture, and tumor xenografts in living animals using molecular imaging. Results show that multitarget antagonization of endogenous miRNAs could be an efficient strategy for targeting metastasis and antiapoptosis in the treatment of metastatic cancer.

Genetically encoded molecular biosensors to image histone methylation in living animals.

Post-translational addition of methyl groups to the amino terminal tails of histone proteins regulates cellular gene expression at various stages of development and the pathogenesis of cellular diseases, including cancer. Several enzymes that modulate these post-translational modifications of histones are promising targets for development of small molecule drugs. However, there is no promising real-time histone methylation detection tool currently available to screen and validate potential small molecule histone methylation modulators in small animal models.

Biotin uptake by mouse and human pancreatic beta cells/islets: a regulated, lipopolysaccharide-sensitive carrier-mediated process.

Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors.

Noninvasive theranostic imaging of HSV1-sr39TK-NTR/GCV-CB1954 dual-prodrug therapy in metastatic lung lesions of MDA-MB-231 triple negative breast cancer in mice.

Metastatic breast cancer is an obdurate cancer type that is not amenable to chemotherapy regimens currently used in clinic. There is a desperate need for alternative therapies to treat this resistant cancer type. Gene-Directed Enzyme Prodrug Therapy (GDEPT) is a superior gene therapy method when compared to chemotherapy and radiotherapy procedures, proven to be effective against many types of cancer in pre-clinical evaluations and clinical trials.

Therapeutic potential of pterostilbene against pancreatic beta-cell apoptosis mediated through Nrf2.

Background And Purpose: Nuclear factor erythroid 2-related factor 2 (Nrf2) is considered to be a 'master regulator' of the antioxidant response as it regulates the expression of several genes including phase II metabolic and antioxidant enzymes and thus plays an important role in preventing oxidative stress-mediated disorders, including diabetes. In this study, for the first time, we investigated the protective properties of a naturally available antioxidant, pterostilbene (PTS), against pancreatic beta-cell apoptosis and the involvement of Nrf2 in its mechanism of action.

Experimental Approach: Immunoblotting and quantitative reverse transcriptase (qRT)-PCR analysis were performed to identify PTS-mediated nuclear translocation of Nrf2 protein and the following activation of target gene expression, respectively, in INS-1E cells.

Therapeutic evaluation of microRNAs by molecular imaging.

MicroRNAs (miRNAs) function as regulatory molecules of gene expression with multifaceted activities that exhibit direct or indirect oncogenic properties, which promote cell proliferation, differentiation, and the development of different types of cancers. Because of their extensive functional involvement in many cellular processes, under both normal and pathological conditions such as various cancers, this class of molecules holds particular interest for cancer research. MiRNAs possess the ability to act as tumor suppressors or oncogenes by regulating the expression of different apoptotic proteins, kinases, oncogenes, and other molecular mechanisms that can cause the onset of tumor development.

Reporter protein complementation imaging assay to screen and study Nrf2 activators in cells and living animals.

NF-E2-related factor-2 (Nrf2) activators promote cellular defense mechanism and facilitate disease prevention associated with oxidative stress. In the present study, Nrf2 activators were identified using cell-based luciferase enzyme fragment complementation (EFC) assay, and the mechanism of Nrf2 activation was studied by molecular imaging. Among the various Nrf2 activators tested, pterostilbene (PTS) showed effective Nrf2 activation, as seen by luminometric screening, and validation in a high throughput-intact cell-imaging platform.