Mor Goldfeder

Theranostic barcoded nanoparticles for personalized cancer medicine

Personalized medicine promises to revolutionize cancer therapy by matching the most effective treatment to the individual patient. Using a nanoparticle-based system, we predict the therapeutic potency of anticancer medicines in a personalized manner. We carry out the diagnostic stage through a multidrug screen performed inside the tumour, extracting drug activity information with single cell sensitivity. By using 100 nm liposomes, loaded with various cancer drugs and corresponding synthetic DNA barcodes, we find a correlation between the cell viability and the drug it was exposed to, according to the matching barcodes. Based on this screen, we devise a treatment protocol for mice bearing triple-negative breast-cancer tumours, and its results confirm the diagnostic prediction. We show that the use of nanotechnology in cancer care is effective for generating personalized treatment protocols.

Influencing the monophenolase/diphenolase activity ratio in tyrosinase.

Tyrosinase is a type 3 copper enzyme with great potential for production of commercially valuable diphenols from monophenols. However, the use of tyrosinase is limited by its further oxidation of diphenols to quinones. We recently determined the structure of the Bacillus megaterium tyrosinase revealing a residue, V218, which we proposed to take part in positioning of substrates within the active site. In the structure of catechol oxidase from Ipomoea batatas, the lack of monophenolase activity was attributed to the presence of F261 near CuA. Consequently, we engineered two variants, V218F and V218G. V218F was expected to have a decreased monophenolase activity, due to the bulky residue extending into the active site. Surprisingly, both V218F and V218G exhibited a 9- and 4.4-fold higher monophenolase/diphenolase activity ratio, respectively. X-ray structures of variant V218F display a flexibility of the phenylalanine residue along with an adjacent histidine, which we propose to be the source of the change in activity ratio.

The unravelling of the complex pattern of tyrosinase inhibition

Tyrosinases are responsible for melanin formation in all life domains. Tyrosinase inhibitors are used for the prevention of severe skin diseases, in skin-whitening creams and to avoid fruit browning, however continued use of many such inhibitors is considered unsafe. In this study we provide conclusive evidence of the inhibition mechanism of two well studied tyrosinase inhibitors, KA (kojic acid) and HQ (hydroquinone), which are extensively used in hyperpigmentation treatment. KA is reported in the literature with contradicting inhibition mechanisms, while HQ is described as both a tyrosinase inhibitor and a substrate. By visualization of KA and HQ in the active site of TyrBm crystals, together with molecular modeling, binding constant analysis and kinetic experiments, we have elucidated their mechanisms of inhibition, which was ambiguous for both inhibitors. We confirm that while KA acts as a mixed inhibitor, HQ can act both as a TyrBm substrate and as an inhibitor.

Mucoadhesive alginate pastes with embedded liposomes for local oral drug delivery

Oral cancers are extremely common among adults with increasing incidences due to human papillomavirus, while treatment modalities are limited. This study aims to develop a new oral mucoadhesive delivery system based on the combination of alginate and liposomes. The polymer provides adhesion properties and induces local release of the drug-loaded carriers, while the liposomes protect the drug from degradation and improve its absorption into the cells. Three hybrid alginate/liposomes delivery systems were investigated: a hybrid paste, which presented excellent adhesive capabilities, yet fast burst release of 90% after 2h; a hybrid hydrogel, demonstrating controllable release rates of 5%, 30% or 60% after 2h but poor mucoadhesive properties. These findings led to the development of a hybrid cross-linked paste. Polymer retention studies demonstrated that 80% of the crosslinked paste was retained on tongue tissue compared to 50% retention of the non-cross-linked pastes, verifying its superior mucoadhesion. The hybrid cross-linked paste presented controllable release rate of 20% after 2h. Alginate paste incorporating doxorubicin loaded liposomes presented similar release rates and were highly effective in promoting cancer cell death. Thus, our innovative formulation, including both desired characteristics of mucoadhesion and sustained liposomes release, is an important milestone in the development of a new potential treatment for oral cancer.

A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

Synthetic Cells Synthesize Therapeutic Proteins inside Tumors

Synthetic cells, artificial cell-like particles, capable of autonomously synthesizing RNA and proteins based on a DNA template, are emerging platforms for studying cellular functions and for revealing the origins-of-life. Here, it is shown for the first time that artificial lipid-based vesicles, containing the molecular machinery necessary for transcription and translation, can be used to synthesize anticancer proteins inside tumors. The synthetic cells are engineered as stand-alone systems, sourcing nutrients from their biological microenvironment to trigger protein synthesis. When pre-loaded with template DNA, amino acids and energy-supplying molecules, up to 2 × 107 copies of green fluorescent protein are synthesized in each synthetic cell. A variety of proteins, having molecular weights reaching 66 kDa and with diagnostic and therapeutic activities, are synthesized inside the particles. Incubating synthetic cells, encoded to secrete Pseudomonas exotoxin A (PE) with 4T1 breast cancer cells in culture, resulted in killing of most of the malignant cells. In mice bearing 4T1 tumors, histological evaluation of the tumor tissue after a local injection of PE-producing particles indicates robust apoptosis. Synthetic cells are new platforms for synthesizing therapeutic proteins on-demand in diseased tissues.

Alginate modified with maleimide-terminated PEG as drug carriers with enhanced mucoadhesion

The goal of this study was to generate a new mucoadhesive carbohydrate-based delivery system composed of alginate (Alg) backbone covalently attached to polyethylene glycol (PEG) modified with a unique functional end-group (maleimide). The immobilization of PEG-maleimide chains significantly improved the mucoadhesion properties attributed to thioether bonds creation via Michael-type addition and hydrogen bonding with the mucus glycoproteins. Mucoadhesion studies using tensile and rotating cylinder assays revealed a 3.6-fold enhanced detachment force and a 2.8-fold enhanced retention time compared to the unmodified polymer, respectively. Additional indirect studies confirmed the presence of polymer-mucus glycoproteins interactions. Drug release experiments were used to evaluate the release profiles from Alg-PEG-maleimide tablets in comparison to Alg and Alg-SH tablets. Viability studies of normal human dermal fibroblasts cells depicted the non-toxic nature of Alg-PEG-maleimide. Overall, our studies disclose that PEG-maleimide substitutions on other biocompatible polymers can lead to the development of useful biomaterials for diverse biomedical applications.

Nanoparticles target early-stage breast cancer metastasis in vivo

Despite advances in cancer therapy, treating cancer after it has metastasized remains an unmet clinical challenge. In this study we demonstrate that 100 nm liposomes target triple-negative murine breast-cancer metastases post intravenous administration. Metastatic breast cancer was induced in BALB/c mice either experimentally, by a tail vein injection of 4T1 cells, or spontaneously, after implanting a primary tumor xenograft. To track their biodistribution in vivo the liposomes were labeled with multi-modal diagnostic agents, including indocyanine green and rhodamine for whole-animal fluorescent imaging, gadolinium for magnetic resonance imaging (MRI), and europium for a quantitative biodistribution analysis. The accumulation of liposomes in the metastases peaked at 24 h post the intravenous administration, similar to the time they peaked in the primary tumor. The efficiency of liposomal targeting to the metastatic tissue exceeded that of a non-liposomal agent by 4.5-fold. Liposomes were detected at very early stages in the metastatic progression, including metastatic lesions smaller than 2 mm in diameter. Surprisingly, while nanoparticles target breast cancer metastasis, they may also be found in elevated levels in the pre-metastatic niche, several days before metastases are visualized by MRI or histologically in the tissue. This study highlights the promise of diagnostic and therapeutic nanoparticles for treating metastatic cancer, possibly even for preventing the onset of the metastatic dissemination by targeting the pre-metastatic niche.

Abstract 2153: Elephant p53 (EP53) expression induces apoptosis of human cancer cells

The goal of our study was to determine if elephant TP53 (EP53) proteins contributing to increased apoptosis and possible cancer resistance in elephants could translate into human cancer cells as a future effective cancer treatment. We previously reported that elephants have a lower than expected rate of cancer, 20 copies of TP53 (1 ancestral gene with introns [EP53-anc] and 19 retrogenes [EP53-retro1-19]), and increased p53-mediated apoptosis induced by DNA damage in elephant cells compared to human cells (Abegglen JAMA 2015). For the current study, we expressed various EP53 proteins in human cancer cells with different p53 status, including osteosarcoma (U2-OS, Saos-2), glioblastoma (T98G), and breast cancer (MCF7). Western blot analysis confirmed EP53 expression. We compared apoptosis in the human cancer cells transfected/transduced with negative control vectors vs. epitope or protein-tagged EP53 exposed to doxorubicin (to induce DNA damage). Apoptosis was measured by cell viability, caspase activity, Propidium Iodide/Annexin V staining, and fluorescence microscopy. We observed a significant increase in caspase activity (normalized to cell viability) of U2-OS and T98G cells expressing EP53 compared to negative control treated cells as shown in Table 1, and apoptosis with p21 restoration in Saos-2. In U2-OS, which overexpress MDM2, EP53 was more effective at inducing apoptosis compared to human TP53. Taken together, we found that EP53-anc restored p53-mediated apoptosis and EP53-anc / EP53-retro9 enhanced p53-mediated apoptosis. These data suggest for the first time that EP53 functions in human cancer cells to promote cell death. Ongoing efforts are exploring the EP53 mechanism of action that leads to increased apoptosis, including expression of EP53 in additional cancer types (lung, melanoma, colon, prostate, and others) with a variety of genetic backgrounds to characterize its functional context. These results support the further exploration of EP53-based cancer therapeutics. Citation Format: Lisa M. Abegglen, Lauren N. Donovan, Genevieve Couldwell, Rosann Robinson, Cristhian Toruno, Mor Goldfeder, Wendy K. Kiso, Dennis L. Schmitt, Aleah F. Caulin, Katrin P. Guillen, Bryan E. Welm, Carlo C. Maley, Avi Schroeder, Joshua D. Schiffman. Elephant p53 (EP53) expression induces apoptosis of human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2153. doi:10.1158/1538-7445.AM2017-2153

Abstract 3686: Elephant TP53 (ep53) retrogene protein expression enhances cellular apoptosis in response to DNA damage

We previously reported that compared with other mammalian species, elephants have a lower than expected rate of cancer (Abegglen JAMA 2015). While exploring cancer resistance in elephants, we discovered that elephants have 20 copies of TP53 (1 ancestral gene with introns and 19 retrogenes) compared to humans with 1 copy. TP53 amplification was associated with increased p53-mediated apoptosis induced by DNA damage in elephant cells compared to human cells. As a follow up to this study, we further explored the function of these elephant TP53 retrogenes by expressing their protein (ep53) in cells with 2 functional TP53 alleles. NIH/3T3 cells were transfected with a mammalian expression construct encoding TP53 retrogene 9 fused to an epitope tag (myc). Western blot analysis probing for myc confirmed expression of retrogene 9. To measure the functional consequence of expression, we compared apoptosis of cells transfected with empty vector to cells transfected with myc-tagged retrogene 9 after doxorubicin treatment. Apoptosis was measured with a triplex assay that assesses viability, cytotoxicity and apoptosis (Apo Tox-Glo, Promega). We observed a significant increase in apoptosis of NIH/3T3 cells expressing retrogene 9 compared to empty vector transfected control cells (10μM doxorubicin, P Citation Format: Lisa M. Abegglen, Rosann Robinson, Lauren Donovan, Aleah F. Caulin, Mor Goldfeder, Avi Schroeder, Carlo C. Maley, Joshua D. Schiffman. Elephant TP53 (ep53) retrogene protein expression enhances cellular apoptosis in response to DNA damage. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3686.