Sharon J. Miller

In Vivo Fluorescence-Based Endoscopic Detection of Colon Dysplasia in the Mouse Using a Novel Peptide Probe

Colorectal cancer (CRC) is a major cause of cancer-related deaths in much of the world. Most CRCs arise from pre-malignant (dysplastic) lesions, such as adenomatous polyps, and current endoscopic screening approaches with white light do not detect all dysplastic lesions. Thus, new strategies to identify such lesions, including non-polypoid lesions, are needed. We aim to identify and validate novel peptides that specifically target dysplastic colonic epithelium in vivo. We used phage display to identify a novel peptide that binds to dysplastic colonic mucosa in vivo in a genetically engineered mouse model of colo-rectal tumorigenesis, based on somatic Apc (adenomatous polyposis coli) gene inactivation. Binding was confirmed using confocal microscopy on biopsied adenomas and excised adenomas incubated with peptide ex vivo. Studies of mice where a mutant Kras allele was somatically activated in the colon to generate hyperplastic epithelium were also performed for comparison. Several rounds of in vivo T7 library biopanning isolated a peptide, QPIHPNNM. The fluorescent-labeled peptide bound to dysplastic lesions on endoscopic analysis. Quantitative assessment revealed the fluorescent-labeled peptide (target/background: 2.17±0.61) binds ∼2-fold greater to the colonic adenomas when compared to the control peptide (target/background: 1.14±0.15), p<0.01. The peptide did not bind to the non-dysplastic (hyperplastic) epithelium of the Kras mice. This work is first to image fluorescence-labeled peptide binding in vivo that is specific towards colonic dysplasia on wide-area surveillance. This finding highlights an innovative strategy for targeted detection to localize pre-malignant lesions that can be generalized to the epithelium of hollow organs.

Targeted detection of murine colonic dysplasia in vivo with flexible multispectral scanning fiber endoscopy

Gastrointestinal cancers are heterogeneous and can overexpress several protein targets that can be imaged simultaneously on endoscopy using multiple molecular probes. We aim to demonstrate a multispectral scanning fiber endoscope for wide-field fluorescence detection of colonic dysplasia. Excitation at 440, 532, and 635 nm is delivered into a single spiral scanning fiber, and fluorescence is collected by a ring of light-collecting optical fibers placed around the instrument periphery. Specific-binding peptides are selected with phage display technology using the CPC;Apc mouse model of spontaneous colonic dysplasia. Validation of peptide specificity is performed on flow cytometry and in vivo endoscopy. The peptides KCCFPAQ, AKPGYLS, and LTTHYKL are selected and labeled with 7-diethylaminocoumarin-3-carboxylic acid (DEAC), 5-carboxytetramethylrhodamine (TAMRA), and CF633, respectively. Separate droplets of KCCFPAQ-DEAC, AKPGYLS-TAMRA, and LTTHYKL-CF633 are distinguished at concentrations of 100 and 1 μM. Separate application of the fluorescent-labeled peptides demonstrate specific binding to colonic adenomas. The average target/background ratios are 1.71 ± 0.19 and 1.67 ± 0.12 for KCCFPAQ-DEAC and AKPGYLS-TAMRA, respectively. Administration of these two peptides together results in distinct binding patterns in the blue and green channels. Specific binding of two or more peptides can be distinguished in vivo using a novel multispectral endoscope to localize colonic dysplasia on real-time wide-field imaging.

In vivo targeting of colonic dysplasia on fluorescence endoscopy with near-infrared octapeptide

Objective To demonstrate a near-infrared (NIR) peptide that is highly specific for colonic adenomas on fluorescence endoscopy in vivo. Design A 3 mm diameter endoscope was adapted to deliver 671 nm illumination and collect NIR fluorescence (696–736 nm). Target (QPIHPNNM) and control (YTTNKH) peptides were labelled with Cy5.5, a NIR dye, and characterised by mass spectra. The peptides were topically administered separately (100 μM) through the endoscopes instrument channel into the distal colon of CPC;Apc mice, genetically engineered to spontaneously develop adenomas. After 5 min for incubation, the unbound peptides were rinsed off, and images were collected at a rate of 10 frames/s. Regions of interest were identified around the adenoma and adjacent normal-appearing mucosa on white light. Intensity measurements were made from these same regions on fluorescence, and the target-to-background ratio (TBR) was calculated. Results An image resolution of 9.8 μm and field of view of 3.6 mm was achieved at a distance of 2.5 mm between the distal end of the instrument and the tissue surface. On mass spectra, the experimental mass-to-charge ratio for the Cy5.5-labelled target and control peptides agreed with expected values. The NIR fluorescence images of adenomas revealed individual dysplastic crypts with distorted morphology. By comparison, only amorphous surface features could be visualised from reflected NIR light. The average TBR for adenomas was found to be 3.42±1.30 and 1.88±0.38 for the target and control peptides, respectively, p=0.007. Conclusion A NIR peptide was shown to be highly specific for colonic adenomas on fluorescence endoscopy in vivo and to achieve sub-cellular resolution images.

Phosphorylation-dependent mineral type specificity for apatite-binding peptide sequences

Apatite-binding peptides discovered by phage display provide an alternative design method for creating functional biomaterials for bone and tooth tissue repair. A limitation of this approach is the absence of display peptide phosphorylation--a post-translational modification important to mineral-binding proteins. To refine the material specificity of a recently identified apatite-binding peptide, and to determine critical design parameters (net charge, charge distribution, amino acid sequence and composition) controlling peptide affinity for mineral, we investigated the effects of phosphorylation and sequence scrambling on peptide adsorption to four different apatites (bone-like mineral, and three types of apatite containing initially 0, 5.6 and 10.5% carbonate). Phosphorylation of the VTKHLNQISQSY peptide (VTK peptide) led to a 10-fold increase in peptide adsorption (compared to nonphosphorylated peptide) to bone-like mineral, and a 2-fold increase in adsorption to the carbonated apatite, but there was no effect of phosphorylation on peptide affinity to pure hydroxyapatite (without carbonate). Sequence scrambling of the nonphosphorylated VTK peptide enhanced its specificity for the bone-like mineral, but scrambled phosphorylated VTK peptide (pVTK) did not significantly alter mineral-binding suggesting that despite the importance of sequence order and/or charge distribution to mineral-binding, the enhanced binding after phosphorylation exceeds any further enhancement by altered sequence order. Osteoblast culture mineralization was dose-dependently inhibited by pVTK and to a significantly lesser extent by scrambled pVTK, while the nonphosphorylated and scrambled forms had no effect, indicating that inhibition of osteoblast mineralization is dependent on both peptide sequence and charge. Computational modeling of peptide-mineral interactions indicated a favorable change in binding energy upon phosphorylation that was unaffected by scrambling. In conclusion, phosphorylation of serine residues increases peptide specificity for bone-like mineral, whose adsorption is determined primarily by sequence composition and net charge as opposed to sequence order. However, sequence order in addition to net charge modulates the mineralization of osteoblast cultures. The ability of such peptides to inhibit mineralization has potential utility in the management of pathologic calcification.

Multispectral endoscopic imaging of colorectal dysplasia in vivo

Light provides a broad range of colors in the visible and near-infrared (400–900 nm) spectrum that can be used to transmit information about molecular expression in normal and diseased tissues. The absorbance spectrum of hemoglobin, the primary chromophore in tissue, is shown in Fig. 1A. Genetic changes that occur in cancer transformation can be heterogeneous, and several signaling pathways may be activated concurrently.1 Moreover, molecular activity levels may vary between individual patients, at different time points, and within the tumor. Thus, a single genetic mutation is unlikely to characterize most disease processes over a general population. Unfortunately, most endoscopic imaging methods are sensitive to only one molecular parameter.2 Thus, a methodology that can image multiple molecular targets simultaneously is better suited to address the heterogeneity of cancer and achieve disease detection with high sensitivity and specificity desired for efficient surveillance. In addition, these multiplexed strategies can potentially visualize the interactions between signaling pathways and allow for better understanding of disease pathogenesis on a systems level. Figure 1 A) The visible and near-infrared spectrum of light provides a broad range of colors to generate molecular images from digestive tract mucosa that can be collected with a B) 1.6 mm diameter multispectral scanning fiber endoscope. C) Excitation is delivered ... Recently, significant progress has been made in advancing new methods of endoscopic imaging to target disease in the digestive tract, including the development of novel instruments and specific contrast agents.3 In this video journal, we aim to demonstrate the application of a novel, multispectral endoscope that uses a scanning fiber to image multiple targets at the same time. Fluorescence imaging can be achieved with high target-to-background ratios, and allow us to see the different target expression patterns in an Apc-mutation dependent mouse model of spontaneous colorectal adenomas using multiple peptides as targeting ligands.4

Targeted imaging of colorectal dysplasia in living mice with fluorescence microendoscopy

We validate specific binding activity of a fluorescence-labeled peptide to colorectal dysplasia in living mice using a miniature, flexible, fiber microendoscope that passes through the instrument channel of an endoscope. The microendoscope delivers excitation light at 473 nm through a fiber-optic bundle with outer diameter of 680 µm to collect en face images at 10 Hz with 4 µm lateral resolution. We applied the FITC-labeled peptide QPIHPNNM topically to colonic mucosa in genetically engineered mice that spontaneously develop adenomas. More than two-fold greater fluorescence intensity was measured from adenomas compared to adjacent normal-appearing mucosa. Images of adenomas showed irregular morphology characteristic of dysplasia.

Detection of colonic inflammation with Fourier transform infrared spectroscopy using a flexible silver halide fiber.

Persistent colonic inflammation increases risk for cancer, but mucosal appearance on conventional endoscopy correlates poorly with histology. Here we demonstrate the use of a flexible silver halide fiber to collect mid-infrared absorption spectra and an interval model to distinguish colitis from normal mucosa in dextran sulfate sodium treated mice. The spectral regime between 950 and 1800 cm−1 was collected from excised colonic specimens and compared with histology. Our model identified 3 sub-ranges that optimize the classification results, and the performance for detecting inflammation resulted in a sensitivity, specificity, accuracy, and positive predictive value of 92%, 88%, 90%, and 88%, respectively.

Dual-functioning phage-derived peptides encourage human bone marrow cell-specific attachment to mineralized biomaterials.

Abstract Cell instructive mineralized biomaterials are a promising alternative to conventional auto-, allo-, and xenograft therapies for the reconstruction of critical sized defects. Extracellular matrix proteins, peptide domains, and functional motifs demonstrating cell and mineral binding activity have been used to improve cell attachment. However, these strategies vary in their tissue regeneration outcomes due to lack of specificity to both regenerative cell populations and the material substrates. In order to mediate cell-specific interactions on apatite surfaces, we identified peptide sequences with high affinity towards apatite (VTKHLNQISQSY, VTK) and clonally derived human bone marrow stromal cells (DPIYALSWSGMA, DPI) using phage display. The primary aims of this study were to measure apatite binding affinity, human bone marrow stromal cell (hBMSC) adhesion strength, and peptide specificity to hBMSCs when the apatite and cell-specific peptides are combined into a dual-functioning peptide. To assess binding affinity to hydroxyapatite (HA), binding isotherms were constructed and peptide binding affinity (K1) determined. HBMSC, MC3T3 and mouse dermal fibroblast (MDF) adhesion strength on biomimetic apatite functionalized with single- and dual-functioning peptide sequences were evaluated using a centrifugation assay. DPI-VTK had the highest binding strength towards hBMSCs (p < 0.01). DPI-VTK, while promoting strong initial attachment to hBMSCs, did not encourage strong adhesions to MC3T3s or fibroblasts (p < 0.01). Taken together, phage display is a promising strategy to identify preferential cell and material binding peptide sequences that can tether specific cell populations onto specific biomaterial chemistries.

Wide-field near-infrared fluorescence endoscope for real-time in vivo imaging

A diode-pumped solid state laser is used to deliver excitation at λex = 671 nm. The beam is expanded by a pair of relay lenses (f1 = 30 and f2 = 50 mm) to 3 mm diameter, filling the aperture of a fluid light cable that is coupled to a Hopkins II rigid endoscope. Near-infrared fluorescence images are collected by the endoscope and transmitted by another set of relay lenses onto a CCD detector that has dimensions of 8.7x6.9 mm2 (1388x1040 pixels). A zoom lens system (F#1.6-16 aperture) with a tunable focal length (20-100 mm) magnifies the image to fill the dimensions of the CCD. A band pass filter allows fluorescence with spectral range λem = 696 to 736 nm to be collected. The system achieves a resolution of 9.8 μm and field-of-view of 3.6 mm at a distance of 2.5 mm between the distal end of the endoscope and the tissue. Images are collected at a rate of 10 frames per second. A filter wheel is incorporated into the handle of the instrument housing to rapidly switch between reflectance and fluorescence images. Cy5.5-labeled peptides were delivered through the 1 mm diameter instrument channel in the endoscope. Near-infrared fluorescence images demonstrated specific peptide binding to spontaneous adenomas that developed beginning at 2 months of age in a genetically-engineered mouse with mutation of one allele in the APC gene. This integrated methodology represents a powerful tool that can achieve real time detection of disease in the colon and other hollow organs.

Fluorescence Detection of Colonic Neoplasia Using a Novel Peptide

Background. Overexpression of AnnexinA2 (ANXA2) has been reported in several epithelial cancers, including colorectal cancers (CRC) (Cancer Letters, 2007). Increasing expression of gastrin gene, and hence progastrin (PG), has also been reported in CRCs. We recently discovered that ANXA2 serves as a non-conventional receptor for PG (Oncogene, 2007). We additionally reported that binding of PGwith extracellular, membrane associated, ANXA2 results in endocytosis and co-localization of PG/ANXA2 intracellularly (Gastro, 2010), and that ANXA2 is required for mediating mitogenic and anti-apoptotic effects of PG on colon cancer cells. ANXA2 is normally involved with intracellular trafficking. During the course of our studies we discovered that ANXA2 is also present on outer membranes of CRC cells, bound to a 29KDa transmembrane protein, and also released into the medium. To further understand physiological significance of these unexpected findings, we examined if ANXA2 is present in serum of patients positive for colorectal growth and if ANXA2 co-localizes with PG in tumor sections, in relation to disease status. Methods, Results and Conclusions. Secretion of ANXA2 (10-50ng/106 cells) was confirmed in conditioned medium (CM) of CRC cells using quantitative Western Blot assay using standard curve with rhANXA2; nontransformed cells were negative. Serum from CRC tumor bearing mice were positive for ANXA2, while control mice were negative. Next, serum samples were obtained from consented patients at time of endoscopy or as discarded samples from CRC patients on day of surgery, as per our IRB protocols. Serum from patients, negative for colonic growths (Normal, N) had low levels of ANXA2 (<1ng/ml), while serum from patients with hyperproliferative growths (Hp), Adenomas (Ad) and adenocarcinomas (AdCA) were on an average positive for 42, 80, and 490 ngs/ml, respectively, suggesting elevation of serum ANXA2 in relation to disease status. Paraffin blocks of Hp, Ads, AdCAs and adjoining N mucosa were obtained as discarded specimens from Department of Pathology and stained immunofluorescently for PG/ANXA2. Hp, Ads and AdCAs were increasingly positive for ANXA2 and PG expression compared to N specimens; ~ 10, 25 and 60 % of respective specimens were positive for intracellular co-localization of PG/ANXA2, suggesting presence of functional PG/ANXA2 axis, confirmed by progressive elevation of nuclear β-catenin and stem cell markers (DCAMKL+1, Lgr5) in relation to disease status. It remains to be determined if, 1) presence of co-localized PG/ANXA2 intracellularly represents a prognostic marker for predicting treatment/recurrence, and 2) if serum ANXA2 levels can be used as a non-invasive diagnostic marker for predicting presence of colorectal growths, stage of disease and/or relapse. Supported by NCI grants CA97959 and CA114264 to PS.