A Bitter Taste Receptor as a Novel Molecular Target on Cancer-Associated Fibroblasts in Pancreatic Ductal Adenocarcinoma

Cancer-associated fibroblasts (CAFs) execute diverse and complex functions in cancer progression. While reprogramming the crosstalk between CAFs and cancer epithelial cells is a promising avenue to evade the adverse effects of stromal depletion, drugs are limited by their suboptimal pharmacokinetics and off-target effects. Thus, there is a need to elucidate CAF-selective cell surface markers that can improve drug delivery and efficacy. Here, functional proteomic pulldown with mass spectrometry was used to identify taste receptor type 2 member 9 (TAS2R9) as a CAF target. TAS2R9 target characterization included binding assays, immunofluorescence, flow cytometry, and database mining. Liposomes conjugated to a TAS2R9-specific peptide were generated, characterized, and compared to naked liposomes in a murine pancreatic xenograft model. Proof-of-concept drug delivery experiments demonstrate that TAS2R9-targeted liposomes bind with high specificity to TAS2R9 recombinant protein and exhibit stromal colocalization in a pancreatic cancer xenograft model. Furthermore, the delivery of a CXCR2 inhibitor by TAS2R9-targeted liposomes significantly reduced cancer cell proliferation and constrained tumor growth through the inhibition of the CXCL-CXCR2 axis. Taken together, TAS2R9 is a novel cell-surface CAF-selective target that can be leveraged to facilitate small-molecule drug delivery to CAFs, paving the way for new stromal therapies

The Development of T7 Phage Based Imaging Probe and the Nature of Floating Cells in Human Embryonic Stem Cell Culture

Target specific nanoparticle based nuclear imaging probes have attracted significant attention recently. In most of the cases, these probes were synthesized by conjugating both affinity reagent and chelator molecules on the particle surface chemically. Subsequently, the isotope ions were attached to the particles via metalchelator interactions. Due to the nature of bio-conjugation process, the number of the chelator molecules and the geometry of the affinity reagent are hard to control. To overcome these limitations, a new approach of constructing an imaging probe was developed. In specific, T7 phage was used as a vehicle to synthesize a particle based imaging probe by displaying peptide that had both metal chelating (6 histidine) and targeting (RGD domains on the phage surface. It was demonstrated that the attachment of copper ions to the metal chelating domain does not interfere with target binding. Furthermore, by reducing the metal ions to metal, we were able to generate a very stable peptide templated metal hybrid T7 phage particle as potential target specific imaging probe. The behavior of the probes against both normal and tumor cells was investigated. The presence of floating cells is a common phenomenon in human embryonic stem (hES) cell cultures. Current assumption for the source of floating cells is that they are from apoptosis / senescence, cellular differentiation and other unavoidable imperfections in culture conditions. Inspired by recent studies on mitotic activities in stem cell colony, we believe the existence of floating cells is resulting from essential events required for hES cells proliferation. It was discovered that not all floating cells are dead and the percentage of floating cells over the attached cells was significantly increased with culture time possibly due to the space limitation for cells in the central part of the colonies. By providing more horizontal or vertical space through colony cutting and overlaid membrane insert in the Z direction, the percentages of floating cells were significantly reduced. All these results indicate that continuous cell division across the colonies is responsible for the emergence of floating cells during hES cell culture.Ph.D. in Biology, May 201

Cholecystokinin-B Receptor-Targeted Nanoparticle for Imaging and Detection of Precancerous Lesions in the Pancreas

Survival from pancreatic cancer remains extremely poor, in part because this malignancy is not diagnosed in the early stages, and precancerous pancreatic intraepithelial neoplasia (PanIN) lesions are not seen on routine radiographic imaging. Since the cholecystokinin-B receptor (CCK-BR) becomes over-expressed in PanIN lesions, it may serve as a target for early detection. We developed a biodegradable fluorescent polyplex nanoparticle (NP) that selectively targets the CCK-BR. The NP was complexed to a fluorescent oligonucleotide with Alexa Fluor 647 for far-red imaging and to an oligonucleotide conjugated to Alexa Fluor 488 for localization by immunohistochemistry. Fluorescence was detected over the pancreas of five- to ten-month-old LSL-KrasG12D/+; P48-Cre (KC) mice only after the injection of the receptor target-specific NP and not after injection of untargeted NP. Ex vivo tissue imaging and selective immunohistochemistry confirmed particle localization only to PanIN lesions in the pancreas and not in other organs, supporting the tissue specificity. A human pancreas tissue microarray demonstrated immunoreactivity for the CCK-BR only in the PanIN lesions and not in normal pancreas tissue. The long-term goal would be to develop this imaging tool for screening human subjects at high risk for pancreatic cancer to enable early cancer detection

Target-Specific Copper Hybrid T7 Phage Particles

Target-specific nanoparticles have attracted significant attention recently, and have greatly impacted life and physical sciences as new agents for imaging, diagnosis, and therapy, as well as building blocks for the assembly of novel complex materials. While most of these particles are synthesized by chemical conjugation of an affinity reagent to polymer or inorganic nanoparticles, we are promoting the use of phage particles as a carrier to host organic or inorganic functional components, as well as to display the affinity reagent on the phage surface, taking advantage of the fact that some phages host well-established vectors for protein expression. An affinity reagent can be structured in a desired geometry on the surface of phage particles, and more importantly, the number of the affinity reagent molecules per phage particle can be precisely controlled. We previously have reported the use of the T7 phage capsid as a template for synthesizing target-specific metal nanoparticles. In this study herein, we reported the synthesis of nanoparticles using an intact T7 phage as a scaffold from which to extend 415 copies of a peptide that contains a hexahistidine (6His) motif for capture of copper ions and staging the conversion of copper ions to copper metal, and a cyclic Arginine-Glycine-Aspartic Acid (RGD4C) motif for targeting integrin and cancer cells. We demonstrated that the recombinant phage could load copper ions under low bulk copper concentrations without interfering with its target specificity. Further reduction of copper ions to copper metal rendered a very stable copper hybrid T7 phage, which prevents the detachment of copper from phage particles and maintains the phage structural integrity even under harsh conditions. Cancer cells (MCF-7) can selectively uptake copper hybrid T7 phage particles through ligand-mediated transmembrane transportation, whereas normal control cells (MCF-12F) uptake 1000-fold less. We further demonstrated that copper hybrid T7 phage could be endocytosed by cancer cells in culture

Summary of CAF screens.

<p>Positive and negative screens were carried out for a cell target, CAFs, and processed with PHASTpep.</p

Normalization strategy and sorting of PHASTpep.

<p>(A) For each screen, the frequencies were divided by the total number of reads of the screen, followed by the frequency of that sequence in the reference library. (B) In order to demonstrate the sorting process, small libraries were created that represented a reference library, two positive screens, and 2 negative screens. For each sequence, a qualitative ranking was determined (predicted ranking) based on the level of frequency assigned in each library. For example, GVTHKLQ was absent in the reference library, high in both positive screens, and absent in both negative screens. Therefore, it was predicted to be ranked very high. Conversely, TPSIYFL was only high in the negative screens and absent elsewhere. Thus it was predicted to rank very low. For each test case (sequence), the predicted ranking was compared to the actual ranking after running the test libraries through our sorting software. R, reference; PS, positive screen; NS, negative screen; A, absent; L, low; H, high.</p

Comparison matrices.

<p>A heat map matrix visualization was generated using conditional formatting in Excel for the top 40 sequences from streptavidin (A) and CAF (B) sets of screens. The first two streptavidin screens used glycine to elute; whereas, the third and forth streptavidin screens were eluted with biotin. Scatter plots compare the frequencies and average frequencies of peptide sequences across independent screen replicates for streptavidin (C) and CAF (D) screens.</p

Validation of PHASTpep software translation and frequency calculations.

<p>(A) The raw data pulled from the fastq file of the Illumina sequencer showing the unique flanking regions (red) surrounding the portion of the DNA sequence corresponding to the displayed peptides (blue). (B) The unique flanking regions were used to isolate the peptide sequences, which were then translated into amino acids. For each sequence, a frequency was calculated corresponding to the number of times it appeared in the run. (C) GUI of the PHASTpep software presented in this paper to automate the data processing and analysis.</p

Summary of Streptavidin screens.

<p>Positive and negative screens were carried out for a protein target, Streptavidin, and processed with PHASTpep.</p

Approach to finding candidate peptide sequences.

<p>(A) The Illumina sequencer outputs fastq files that are separated by barcodes. For each of these files, the portion of DNA corresponding to the displayed peptides was isolated and translated. The number of times each sequence was read in a run was summed to obtain the frequency associated with that sequence, which was subsequently divided by the total number of reads from the run and then by the frequency of that sequence in the reference library. This processing resulted in a normalized frequency for each sequence of a run. (B) Sequences present in one screen but absent in another were set to the non-zero mode of the absent screen rather than zero to prevent later division by zero. The normalized frequencies across all positive screens were averaged as well as across all negative screens. The average positive normalized frequency was divided by the average negative normalized frequency and this ratio was used to sort the sequences so that sequences high across positive screens and low across negative screens distilled to the top fraction. Sequences ordered by ratio created the rows of the comparison matrix showing all of the normalized frequencies for each sequence across all screens, facilitating identification of the most selective sequences. * PhD libraries from NEB are generated with constrained codons. When using this library, sequences containing codons not represented in the library are removed.</p