carbocyanines and Colonic-Neoplasms

Topics: Adenoma; Animals; Carbocyanines; Cell Line, Tumor; Colonic Neoplasms; Colonoscopy; Fluorescent Dyes; HT29 Cells; Humans; Image-Guided Biopsy; Infrared Rays; Intestinal Mucosa; Ligands; Mice; NIH 3T3 Cells; Peptides; Protein Binding; Proto-Oncogene Proteins c-met; Sensitivity and Specificity

Legumain is one of the cysteine proteases which can serve as an essential indicator for cancer diagnosis. Near-infrared (NIR) nanoprobes with fluorescence "Turn On" property are advantageous in cancer diagnosis. However, to the best of our knowledge, using a completely organic NIR nanoprobe to image legumain activity either in vitro or in vivo has not been reported. Herein, employing a CBT-Cys click condensation reaction, we used a rationally designed NIR probe Cys(StBu)-Ala-Ala-Asn-Lys(Cy5.5)-CBT (1) to synthesize its nanoprobes 1-NPs with self-quenched fluorescence. Cell and animal experiments indicated that our nanoprobes were able to specifically image legumain activity in living cells and tumors with a NIR fluorescence "Turn On" manner. We envision that the nanoprobes could be applied for the diagnosis of legumain-related diseases in the near future.

Topics: Animals; Carbocyanines; Click Chemistry; Colonic Neoplasms; Cysteine Endopeptidases; Fluorescent Dyes; HCT116 Cells; Humans; Infrared Rays; Mice; Microscopy, Fluorescence; Oligopeptides; Optical Imaging

Photodynamic therapy (PDT) of cancer is dependent on three primary components: photosensitizer (PS), light and oxygen. Because these components are interdependent and vary during the dynamic process of PDT, assessing PDT efficacy may not be trivial. Therefore, it has become necessary to develop pre-treatment planning, on-line monitoring and dosimetry strategies during PDT, which become more critical for two or more chromophore systems, for example, PS-CD (Photosensitizer-Cyanine dye) conjugates developed in our laboratory for fluorescence-imaging and PDT of cancer. In this study, we observed a significant impact of variable light dosimetry; (i) high light fluence and fluence rate (light dose: 135 J/cm², fluence rate: 75 mW/cm²) and (ii) low light fluence and fluence rate (128 J/cm² and 14 mW/cm² and 128 J/cm² and 7 mW/cm²) in photobleaching of the individual chromophores of PS-CD conjugates and their long-term tumor response. The fluorescence at the near-infrared (NIR) region of the PS-NIR fluorophore conjugate was assessed intermittently via fluorescence imaging. The loss of fluorescence, photobleaching, caused by singlet oxygen from the PS was mapped continuously during PDT. The tumor responses (BALB/c mice bearing Colon26 tumors) were assessed after PDT by measuring tumor sizes daily. Our results showed distinctive photobleaching kinetics rates between the PS and CD. Interestingly, compared to higher light fluence, the tumors exposed at low light fluence showed reduced photobleaching and enhanced long-term PDT efficacy. The presence of NIR fluorophore in PS-CD conjugates provides an opportunity of fluorescence imaging and monitoring the photobleaching rate of the CD moiety for large and deeply seated tumors and assessing PDT tumor response in real-time.

Topics: Animals; Carbocyanines; Chlorophyll; Colonic Neoplasms; Dose-Response Relationship, Radiation; Fluorescent Dyes; Glycoconjugates; Indoles; Infrared Rays; Mice; Mice, Inbred BALB C; Optical Imaging; Photobleaching; Photochemotherapy; Photosensitizing Agents; Propionates; Radiometry; Singlet Oxygen; Spectrometry, Fluorescence; Xenograft Model Antitumor Assays

We report the development, characterization, and validation of a peptide specific for the extracellular domain of HER2. This probe chemistry was developed for molecular imaging by using a structural model to select an optimal combination of amino acids that maximize the likelihood for unique hydrophobic and hydrophilic interactions with HER2 domain 3. The sequence KSPNPRF was identified and conjugated with either FITC or Cy5.5 via a GGGSK linker using Fmoc-mediated solid-phase synthesis to demonstrate flexibility for this chemical structure to be labeled with different fluorophores. A scrambled sequence was developed for control by altering the conformationally rigid spacer and moving both hydrophobic and hydrophilic amino acids on the C-terminus. We validated peptide specificity for HER2 in knockdown and competition experiments using human colorectal cancer cells in vitro, and measured a binding affinity of kd = 21 nM and time constant of k = 0.14 min(-1) (7.14 min). We used this peptide with either topical or intravenous administration in a preclinical model of colorectal cancer to demonstrate specific uptake in spontaneous adenomas and to show feasibility for real time in vivo imaging with near-infrared fluorescence. We used this peptide in immunofluorescence studies of human proximal colon specimens to evaluate specificity for sessile serrated and sporadic adenomas. Improved visualization can be used endoscopically to guide tissue biopsy and detect premalignant lesions that would otherwise be missed. Our peptide design for specificity to HER2 is promising for clinical translation in molecular imaging methods for early cancer detection.

Topics: Animals; Carbocyanines; Cell Line, Tumor; Colon; Colonic Neoplasms; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Mice; Microscopy, Fluorescence; Molecular Imaging; Peptides; Receptor, ErbB-2; Solid-Phase Synthesis Techniques

Fluorescence molecular imaging can be employed for the development of novel cancer targeting agents. Herein, we investigated the pharmacokinetics (PK) and cellular uptake of Dmt-Tic-Cy5, a delta-opioid receptor (δOR) antagonist-fluorescent dye conjugate, as a tumor-targeting molecular imaging agent. δOR expression is observed normally in the CNS, and pathologically in some tumors, including lung liver and breast cancers. In vitro, in vivo, and ex vivo experiments were conducted to image and quantify the fluorescence signal associated with Dmt-Tic-Cy5 over time using in vitro and intravital fluorescence microscopy and small animal fluorescence imaging of tumor-bearing mice. We observed specific retention of Dmt-Tic-Cy5 in tumors with maximum uptake in δOR-expressing positive tumors at 3 h and observable persistence for >96 h; clearance from δOR nonexpressing negative tumors by 6 h; and systemic clearance from normal organs by 24 h. Live-cell and intravital fluorescence microscopy demonstrated that Dmt-Tic-Cy5 had sustained cell-surface binding lasting at least 24 h with gradual internalization over the initial 6 h following administration. Dmt-Tic-Cy5 is a δOR-targeted agent that exhibits long-lasting and specific signal in δOR-expressing tumors, is rapidly cleared from systemic circulation, and is not retained in non-δOR-expressing tissues. Hence, Dmt-Tic-Cy5 has potential as a fluorescent tumor imaging agent.

Topics: Animals; Apoptosis; Carbocyanines; Cell Proliferation; Colonic Neoplasms; Dipeptides; Female; Fluorescent Dyes; Humans; Immunoenzyme Techniques; Kinetics; Mice; Mice, Nude; Narcotic Antagonists; Real-Time Polymerase Chain Reaction; Receptors, Opioid, delta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectroscopy, Near-Infrared; Tetrahydroisoquinolines; Tissue Distribution; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

To enable multimodal in vivo and ex vivo optical imaging of the biodistribution and tumor accumulation of core-crosslinked polymeric micelles (CCPMs).. mPEG-b-p(HPMAm-Lac)-based polymeric micelles, core-crosslinked via cystamine and covalently labeled with two different fluorophores (Dy-676/488), were synthesized. The CCPMs were intravenously injected into CT26 tumor-bearing mice.. Upon intravenous injection, the CCPMs accumulated in CT26 tumors reasonably efficiently, with values reaching approximately 4%ID at 24 h. Ex vivo two-photon laser scanning microscopy confirmed efficient extravasation of the image-guided CCPMs out of tumor blood vessels and relatively deep penetration into the tumor interstitium.. CCPMs were labeled with multiple fluorophores, and the results obtained exemplify that combining several different in vivo and ex vivo optical imaging techniques is highly useful for analyzing the biodistribution and tumor accumulation of nanomedicines.

Topics: Acrylamides; Animals; Carbocyanines; Cell Line, Tumor; Colon; Colonic Neoplasms; Drug Carriers; Drug Delivery Systems; Fluorescent Dyes; Humans; Indoles; Mice; Mice, Nude; Micelles; Multimodal Imaging; Optical Imaging; Polyethylene Glycols; Tissue Distribution

Overexpression of neurotensin receptors (NTRs) has been suggested to play important roles in the growth and survival of a variety of tumor types. The aim of this study is to develop a dual-modality probe (64Cu -DOTA-NT-Cy5.5) for imaging NTR1 expression in vivo with both positron emission tomography (PET) and fluorescence. In this approach, the thiol group and N terminal amino group of neurotensin analogue (Cys-NT) were chemically modified with Cy5.5 dye and DOTA chelator, respectively. After radiolabeling with 64Cu, the resulting probe (64Cu-DOTA-NT-Cy5.5) was evaluated in NTR1 positive HT-29 tumor model. Small animal PET quantification analysis demonstrated that the tumor uptake was 1.91±0.22 and 1.79±0.16%ID/g at 1 and 4 h postinjection (p.i.), respectively. The tumor-to-muscle ratio was 17.44±3.25 at 4 h p.i. based on biodistribution. Receptor specificity was confirmed by the successful blocking experiment at 4 h p.i. (0.42±0.05%ID/g). In parallel with PET experiment, fluorescence imaging was also performed, which demonstrated prominent tumor uptake in HT-29 model. As a proof of concept, an imaging guided surgery was performed to the fluorescent moiety of this probe and could provide potential surgery guidance for NTR positive patients. In summary, our results clearly indicated that the dual-modality probe, 64Cu-DOTA-NT-Cy5.5, could serve as a promising agent to image NTR positive tumors in vivo.

Topics: Animals; Carbocyanines; Chelating Agents; Colonic Neoplasms; Copper Radioisotopes; Fluorescent Dyes; Heterocyclic Compounds, 1-Ring; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Positron-Emission Tomography; Radiopharmaceuticals; Receptors, Neurotensin; Tissue Distribution; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The aim of this study was to evaluate a new fluorescently labeled chimeric anti-CEA antibody for improved detection and resection of colon cancer.. Frozen tumor and normal human tissue samples were stained with chimeric and mouse antibody-fluorophore conjugates for comparison. Mice with patient-derived orthotopic xenografts (PDOX) of colon cancer underwent fluorescence-guided surgery (FGS) or bright-light surgery (BLS) 24 hr after tail vein injection of fluorophore-conjugated chimeric anti-CEA antibody. Resection completeness was assessed using postoperative images. Mice were followed for 6 months for recurrence.. The fluorophore conjugation efficiency (dye/mole ratio) improved from 3-4 to >5.5 with the chimeric CEA antibody compared to mouse anti-CEA antibody. CEA-expressing tumors labeled with chimeric CEA antibody provided a brighter fluorescence signal on frozen human tumor tissues (P = 0.046) and demonstrated consistently lower fluorescence signals in normal human tissues compared to mouse antibody. Chimeric CEA antibody accurately labeled PDOX colon cancer in nude mice, enabling improved detection of tumor margins for more effective FGS. The R0 resection rate increased from 86% to 96% with FGS compared to BLS.. Improved conjugating efficiency and labeling with chimeric fluorophore-conjugated antibody resulted in better detection and resection of human colon cancer in an orthotopic mouse model.

Topics: Animals; Antibodies, Monoclonal; Carbocyanines; Carcinoembryonic Antigen; Chimera; Colonic Neoplasms; Diagnostic Imaging; Fluorescent Antibody Technique; Fluorescent Dyes; Heterografts; Humans; Image Enhancement; Mice; Mice, Nude

Non human antibodies administered to human patients often generate anti-antibody responses, leading in extreme cases to anaphylactic shock. Completely human antibodies are therefore favored over their murine, chimeric and humanized counterparts. However, the accurate evaluation of human antibodies on human tissue samples cannot be achieved using indirect immunohistochemical methods because of endogenous immunoglobulins that are co-detected by the secondary antibodies. Direct detection is often used instead, but this lacks the signal amplification conferred by the secondary antibody and is therefore less sensitive. We developed a simple fluorescence-based indirect immunohistochemical method that allows human primary antibodies bound specifically to their target antigens in human tissue samples to be detected clearly and without interfering background staining. This approach involves a biotinylated human primary antibody (H10(Biotin)) and Cy3-conjugated streptavidin (Strep(Cy3)). We tested the protocol using a human carcinoembryonic antigen (CEA) specific IgG1 (H10). We identified an exposure time threshold that allowed the elimination of low Strep(Cy3) background staining, yet achieved sufficient signal amplification to make our approach four times more sensitive than comparable direct immunohistochemical procedures. The principle of this indirect immunohistochemical assay should be transferable to other species allowing the specific and sensitive detection of any primary antibody on homologous tissues.

Topics: Animals; Antibodies, Monoclonal; Biotin; Biotinylation; Carbocyanines; Carcinoembryonic Antigen; Carcinoma; CHO Cells; Colonic Neoplasms; Cricetulus; Fluorescent Antibody Technique, Indirect; HEK293 Cells; Humans; Immunoglobulin G; Mice; Sensitivity and Specificity; Staining and Labeling; Streptavidin

Receptor targeted therapy is advantageous in overcoming the toxicity burden of conventional cancer chemotherapeutics. Over expression of epidermal growth factor receptor (EGFR) on cancer cells and its role in metastasis, malignancy and drug resistance in many human cancers lead to its selection as a promising target for cancer treatment. The present work investigated the preparation and characterization of docetaxel (DTXL) loaded gamma-poly (glutamic acid) (gamma-PGA) nanoparticles (Nps) conjugated with EGFR antibody (Cetuximab, CET) targeted to colon cancer cells (HT-29), highly over expressing EGFR. The flow cytometric analysis revealed two fold increased cellular uptake of CET-DTXL-gamma-PGA Nps by HT-29 (EGFR +ve) cells compared to that of IEC-6 (EGFR-ve) cells confirming the active targeting. Cytotoxicity assays (MTT and LDH) showed superior anti-proliferative activity of CET-DTXL-gamma-PGA NPs over DTXL-gamma-PGA Nps against HT-29 cells. The cell cycle analysis indicated that CET-DTXL-gamma-PGA NPs induced cell death in enhanced percentage of HT-29 cells by undergoing cell cycle arrest in G2/M phase compared to that of DTXL-gamma-PGA Nps. The mechanism of cancer cell death was analyzed via apoptotic and mitochondrial membrane potential assays and showed that targeted Nps treatment reduced the mitochondrial membrane potential thereby inducing enhanced HT-29 cell death (apoptosis and necrosis). The biodistribution of targeted and non-targeted Nps were analyzed in vivo in Swiss albino mice using NIR imaging. ICG-CET-DTXL-gamma-PGA Nps (targeted) and ICG-DTXL-gamma-PGA Nps (non-targeted) followed the similar biodistribution pattern in vivo, but with different elimination time. In short, CET-DTXL-gamma-PGA nanoparticles enhance the tumor selective therapeutic efficacy for colon cancer.

Topics: Animals; Annexins; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Benzimidazoles; Carbocyanines; Cell Cycle; Cell Death; Cetuximab; Colonic Neoplasms; Docetaxel; Drug Carriers; ErbB Receptors; Hemolysis; HT29 Cells; Humans; Mice; Nanomedicine; Nanoparticles; Polyglutamic Acid; Taxoids; Tissue Distribution

This study aimed to evaluate the influence of phenothiazine derivatives (PDs) on the intracellular accumulation of cyanine dye DiOC6(3) in doxorubicin-resistant LoVo-DX cell line, with overexpression of P-glycoprotein.. In order to maintain a high expression level of P-gp, the LoVo-DX cells were grown in the presence of doxorubicin (100 ng/ml). The time-dependent fluorescence signal (T-DFS) of the intracellular accumulation of DiOC6(3), in the presence of PDs, was then recorded. The rate constants k1, k2, k3 and amplitudes of T-DFS, describing the intracellular accumulation process, were determined based on the respective theoretical equation.. The values of k1 and k2 were dependent on the hydrophobicity (logP) of the PDs used as drug resistance modulators. A rise of k1 and k2 values was observed when the logP of PDs increased.. We suggest that the k1 and k2 rate constants could be regarded as useful parameters for assessment of PDs as well as of other compounds of potential application as reversers of multidrug resistance.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Carbocyanines; Colonic Neoplasms; Doxorubicin; Drug Resistance, Neoplasm; Fluorescent Dyes; Microscopy, Confocal; Phenothiazines; Spectrometry, Fluorescence; Tumor Cells, Cultured

In recent years, polymer drug carriers based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with pH-triggered drug release have shown enhanced uptake in solid tumors and excellent antitumor activity. Here, the impact of the structure of the acid-labile spacer between the drug and the polymer carrier on the biodistribution of both the drug and the carrier was studied using in vivo noninvasive multispectral optical imaging of dual fluorescently labeled HPMA copolymers. Five different spacers containing a pH-sensitive hydrazone bond were synthesized and used to combine a fluorescent model drug with a polymer backbone, conjugated with another non-releasable fluorescent dye. Two copolymers differing in polymer chain structure (linear and star-like) and molecular weight (30 and 200kDa) were used to distinguish between carriers with molecular weights above and below the limit for renal filtration. The rate of model drug release from the conjugates was determined in vitro. The biodistributions of the six most promising conjugates were investigated in vivo in athymic nude mice inoculated with human colon carcinoma xenograft. The structure of the spacer in the vicinity of the hydrazone bond significantly influenced the release rate of the model drug. The slow release rate of a pyridyl group bearing spacer resulted in a greater amount of the model drug being transported to the tumor, which was independent of the carrier structure. The results of this study emphasize the importance of careful selection of the structure and appropriate spacer when designing polymer conjugates intended for passive tumor targeting.

Topics: Acrylamides; Animals; Carbocyanines; Cell Line, Tumor; Colonic Neoplasms; Delayed-Action Preparations; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Indoles; Male; Mice; Mice, Nude; Models, Molecular; Tissue Distribution

To demonstrate a near-infrared (NIR) peptide that is highly specific for colonic adenomas on fluorescence endoscopy in vivo.. 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 endoscope's 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.. 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.. A NIR peptide was shown to be highly specific for colonic adenomas on fluorescence endoscopy in vivo and to achieve sub-cellular resolution images.

Topics: Adenoma; Animals; Biopsy; Carbocyanines; Colonic Neoplasms; Diagnostic Imaging; Endoscopy; Fluorescence; Humans; Infrared Rays; Mice; Oligopeptides; Sensitivity and Specificity; Staining and Labeling

Preclinical in vivo characterization of new polymeric drug conjugate candidates is crucial for understanding the effects of certain chemical modifications on distribution and elimination of these carrier systems, which is the basis for rational drug design. In our study we synthesized dual fluorescent HPMA copolymers of different architectures and molecular weights, containing one fluorescent dye coupled via a stable hydrazide bond functioning as the carrier label and the other one modeling the drug bound to a carrier via a pH-sensitive hydrolytically cleavable hydrazone bond. Thus, it was possible to track the in vivo fate, namely distribution, elimination and tumor accumulation, of the polymer drug carrier and a cleavable model drug simultaneously and noninvasively in nude mice using multispectral optical imaging. We confirmed our in vivo results by more detailed ex vivo characterization (imaging and microscopy) of autopsied organs and tumors. There was no significant difference in relative biodistribution in the body between the 30 KDa linear and 200 KDa star-like polymer, but the star-like polymer circulated much longer. We observed a moderate accumulation of the polymeric carriers in the tumors. The accumulation of the pH-sensitive releasable model drug was even higher compared to the polymer accumulation. Additionally, we were able to follow the long-term in vivo fate and to prove a time-dependent tumor accumulation of HPMA copolymers over several days.

Topics: Animals; Antineoplastic Agents; Benzopyrans; Carbocyanines; Cell Line, Tumor; Colonic Neoplasms; Delayed-Action Preparations; Drug Carriers; Drug Delivery Systems; Fluorescent Dyes; Hydrogen-Ion Concentration; Indoles; Methacrylates; Mice; Mice, Nude; Polymers; Tissue Distribution

The purpose of this study was to use a near-infrared (NIR) fluorescent cyclic His-Try-Gly-Phe peptide to characterize and image the expressions of matrix metalloproteinases (MMPs), which are correlated with cancer promotion, in an inflammation-induced colorectal cancer (ICRC) model. We explored the relationship between the development of colon cancer and the expression of MMPs at the same colonic sites in ICRC models. To develop ICRC models, mice were administered a single intraperitoneal dose (10 mg/kg) of azoxymethane (AOM) and exposed orally to 2% dextran sodium sulfate (DSS) for one week. MMP-2 expression and β-catenin activation in colonic lesions were characterized by immunohistochemical (IHC) staining. After being treated with inducers for some time, cancerous lesions were found to express high β-catenin and MMP-2. The profiles of MMP expression were correlated with β-catenin activation in the colonic lesions. c(KAHWGFTLD)NH(2) (C6) peptide was prepared by standard Fmoc peptide synthesis to target MMPs. Molecular weight of Cy5.5-C6 was 1,954.78 g/mol (calculated MW = 1955.23 g/mol). The in vitro characterization of Cy5.5-C6 showed MMP binding specificity in a cell experiment. In vivo NIRF imaging showed high accumulation of Cy5.5-C6 in tumors with associated expression of MMP-2 in colonic lesions after intravenous injection. The MMP-2 specificity of Cy5.5-C6 was confirmed by successful inhibition of probe uptake in the tumor due to the presence of excess C6 peptide. The use of Cy5.5-C6 to target MMP-2 has the potential to be developed into an effective molecular imaging agent to monitor ICRC progress.

Topics: Animals; Azoxymethane; beta Catenin; Blotting, Western; Carbocyanines; Carcinogens; Colonic Neoplasms; Dextran Sulfate; Diagnostic Imaging; Disease Models, Animal; Disease Progression; Immunoenzyme Techniques; Inflammation; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Peptide Fragments

Fluorescent-labeled peptides are being developed to improve the endoscopic detection of colonic dysplasia.. To demonstrate a near-infrared peptide multimer that functions as a phage mimic for in vivo detection of colonic adenomas.. A peptide multimer was synthesized by using trilysine as a dendritic wedge to mimic the presentation of peptides on phage, and all peptides, including the multimer, were fluorescent-labeled with Cy5.5.. Small-animal imaging facility. ANIMAL SUBJECTS: Genetically engineered CPC;Apc mice that spontaneously develop colonic adenomas.. Near-infrared-labeled AKPGYLS peptide multimer was administered topically into the distal colons of the mice, and endoscopic images of adenomas were captured. Fluorescence intensities were quantified by target-to-background (T/B) ratios, and adenoma dimensions were measured with calipers after imaging. Validation of specific peptide binding was performed on cryosectioned specimens and cells by using confocal microscopy and flow cytometry.. Fluorescence T/B ratios from colonic adenomas and adjacent normal-appearing mucosa.. AKP-multimer, monomer, trilysine core, and Cy5.5 resulted in mean (± SD) T/B ratios of 3.85 ± 0.25, 2.21 ± 0.13, 1.56 ± 0.12, and 1.19 ± 0.11, respectively, P < .01 on in vivo imaging. Peptide multimer showed higher contrast and greater specificity for dysplastic crypts as compared with other probes. Peptide multimer demonstrated significantly greater binding to HT29 cells on flow cytometry and fluorescence microscopy in comparison to monomer and trilysine core. A binding affinity of 6.4 nm/L and time constant of 0.1136 minutes(-1) (8.8 minutes) was measured for multimer.. Only distal colonic adenomas were imaged.. Peptide multimers combine strengths of multiple individual peptides to enhance binding interactions and demonstrate significantly higher specificity and affinity for tumor targets.

Topics: Adenoma; Amino Acid Sequence; Animals; Bacteriophages; Carbocyanines; Chemistry Techniques, Synthetic; Colonic Neoplasms; Colonoscopy; Flow Cytometry; Fluorescent Dyes; HT29 Cells; Humans; Mice; Mice, Transgenic; Molecular Imaging; Optical Imaging; Peptide Library; Peptides

To prospectively depict carcinoembryonic antigen (CEA)-expressing tumors in mice with a high-affinity probe consisting of a near-infrared (NIR) fluorochrome and the clinically used anti-CEA antibody fragment arcitumomab.. This study was approved by the regional animal committee. By coupling a NIR fluorescent (NIRF) cyanine dye (DY-676) to a specific antibody fragment directed against CEA (arcitumomab) and a nonspecific IgG Fab fragment, a bio-optical high-affinity fluorescent probe (anti-CEA-DY-676) and a low-affinity fluorescent probe (FabIgG-DY-676) were designed. The dye-to-protein ratios were determined, and both probes were tested for NIRF imaging in vitro on CEA-expressing LS-174T human colonic adenocarcinoma cells and CEA-nonexpressing A-375 human melanoma cells by using a bio-optical NIR small-animal imager. In vivo data of xenografted LS-174T and A-375 tumors in mice (n = 10) were recorded and statistically analyzed (Student t test).. The dye-to-protein ratios were determined as 3.0-3.5 for both probes. In vitro experiments revealed the specific binding of the anti-CEA-DY-676 probe on CEA-expressing cells as compared with CEA-nonexpressing cells; the FabIgG-DY-676 probe showed a markedly lower binding affinity to cells. In vivo LS-174T tumors xenografted in all mice could be significantly distinguished from A-375 tumors with application of the anti-CEA-DY-676 but not with that of the FabIgG-DY-676 at different times (2-24 hours, P < .005) after intravenous injection of the probes. Semiquantitative analysis revealed maximal fluorescence signals of anti-CEA-DY-676 to CEA-expressing tumors about 8 hours after injection.. Findings of this study indicate the potential use of the high-affinity probe anti-CEA-DY-676 for specific NIRF imaging in in vivo tumor diagnosis.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Carbocyanines; Carcinoembryonic Antigen; Colonic Neoplasms; Female; Immunoglobulin Fab Fragments; Mice; Mice, Inbred BALB C; Polymerase Chain Reaction; Prospective Studies; Spectrometry, Fluorescence; Spectrophotometry, Infrared; Whole Body Imaging

The peripheral benzodiazepine receptor (PBR) is a trans-mitochondrial membrane protein that modulates steroid biosynthesis. Recently, up-regulation and nuclear localization of PBR has been shown to be associated with colon, prostate, and breast cancer. PBR has been targeted by the exogenous synthetic ligand, PK11195, for various purposes including imaging. To capitalize on these observations, we developed a high-throughput, noninvasive, in vivo imaging approach to detect spontaneously arising colonic tumors in mice using a novel PBR-targeted molecular imaging agent (NIR-conPK11195). NIR-conPK11195 localized and was retained in colonic adenomas and carcinomas in Smad3(-/-) mice but not in non-neoplastic hamartomas or chronically inflamed colonic tissue. Using a fluorescence signal-to-noise ratio of > or =4-fold 13 h after injection of the agent, we detected colonic tumors with a sensitivity of 67% and a specificity of 86% in a cohort of 37 Smad3(-/-) mice and control littermates. Furthermore, using oral administration of dextran sulfate to induce colonic inflammation, we showed that the clearance profile of NIR-conPK11195 distinguished transient uptake in inflammatory tissue from longer term retention in tumors. Taken together, these results indicate that NIR-conPK11195 is a promising optical molecular imaging tool to rapidly screen for colonic tumors in mice and to discriminate inflammation from cancer.

Topics: Animals; Biomarkers, Tumor; Carbocyanines; Colonic Neoplasms; Diagnosis, Differential; Fluorescent Dyes; Inflammation; Isoquinolines; Mice; Mice, Knockout; Molecular Structure; Receptors, GABA-A; Smad3 Protein; Spectroscopy, Near-Infrared; Staining and Labeling

We constructed a novel database of the proteome of DLD-1 colon cancer cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of fluorescence-labeled proteins followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis. The database consists of 258 functionally categorized proteins corresponding to 314 protein spots. The majority of the proteins are oxidoreductases, cytoskeletal proteins and nucleic acid binding proteins. Phosphatase treatment showed that 28% of the protein spots on the gel are phosphorylated, and mass spectrometric analysis identified 21 of them. Proteins of DLD-1 cells and of laser-microdissected colon cancer tissues showed similar distribution on 2D gels, suggesting the utility of our database for clinical proteomics.

Topics: Amino Acid Sequence; Carbocyanines; Cell Line, Tumor; Colonic Neoplasms; Databases, Protein; Electrophoresis, Gel, Two-Dimensional; Humans; Molecular Sequence Data; Phosphoproteins; Phosphoric Monoester Hydrolases; Proteins; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Response to photodynamic therapy depends on adequate tumor oxygenation as well as sufficient accumulation of photosensitizer in the tumor. The goal of this study was to investigate the presence of hypoxia and retention of the photosensitizer Photofrin in the tumors of patients with intra-abdominal carcinomatosis or sarcomatosis.. Tumor nodules from 10 patients were studied. In nine of these patients, hypoxia was identified in histological sections of biopsied tumor after administration of the hypoxia marker 2-(2-nitroimidazol-1[H]-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5). In separate tumor nodules from 10 patients, Photofrin uptake was measured by fluorescence after tissue solubilization.. Hypoxia existed in the tumors of five patients, with three of these patients demonstrating at least one severely hypoxic nodule. Physiological levels of oxygen were present in the tumors of four patients. An association between tumor size and hypoxia was not evident because some tumor nodules as small as approximately 2 mm in diameter were severely hypoxic. However, even these tumor nodules contained vascular networks. Three patients with severely hypoxic tumor nodules exhibited moderate levels of Photofrin uptake of 3.9 +/- 0.4 to 3.9 +/- 0.5 ng/mg (mean +/- SE). The four patients with tumors of physiological oxygenation did not consistently exhibit high tumor concentrations of Photofrin: mean +/- SE drug uptake among these patients ranged from 0.6 +/- 0.8 to 5.8 +/- 0.5 ng/mg.. Carcinomatosis or sarcomatosis of the i.p. cavity may exhibit severe tumor hypoxia. Photofrin accumulation in tumors varied by a factor of approximately 10x among all patients, and, on average, those with severe hypoxia in at least one nodule did not demonstrate poor Photofrin uptake in separate tumor samples. These data emphasize the need for reconsideration of the generally accepted paradigm of small tumor size, good oxygenation, and good drug delivery because this may vary on an individual tumor basis.

Topics: Appendiceal Neoplasms; Benzimidazoles; Binding, Competitive; Carbocyanines; Colonic Neoplasms; Dihematoporphyrin Ether; Etanidazole; Female; Gastrointestinal Neoplasms; Gastrointestinal Stromal Tumors; Humans; Hydrocarbons, Fluorinated; In Vitro Techniques; Intestine, Small; Male; Microscopy, Fluorescence; Ovarian Neoplasms; Oxygen; Photochemotherapy; Sarcoma

Macromolecules accumulate in solid tumors and can thus be used as carriers for the delivery of attached contrast agents to tumors. We report the synthesis and use of serum protein-dye conjugates consisting of transferrin (Tf) or human serum albumin (HSA) and an indotricarbocyanine (ITCC) derivative as contrast agents for the optical imaging of tumors. The compounds were characterized with respect to their photophysical properties and tested in vitro for their ability to bind to tumor cells and in vivo for their potential to delineate experimental tumors. In contrast to HAS-ITTC, Tf-ITCC showed receptor-mediated uptake by HT29 human colon cancer cells in vitro. After intravenous injection into HT29 tumor-bearing nude mice both compounds induced increased fluorescence contrast of tumors in vivo. After 24 h the contrast between tumor and normal tissue was significantly higher for Tf-ITCC than for HAS-ITCC. Dye-induced fluorescence was found to be predominantly located in perinecrotic areas of the tumor. Furthermore, Tf-ITCC produced fluorescence of viable tumor cells, whereas HAS-ITCC fluorescence was recorded along connective tissue. We conclude that ITCC-labeled Tf and HSA can serve as macromolecular contrast agents for the optical imaging of tumors, with Tf-ITCC showing higher efficiency.

Topics: Animals; Carbocyanines; Colonic Neoplasms; Contrast Media; Fluorescent Dyes; Humans; Macromolecular Substances; Mice; Mice, Nude; Serum Albumin; Transferrin

Colorectal cancer frequently disseminates through the portal vein into the liver. In this study, outbred Swiss nude mice were adapted to facilitate the induction of liver metastases by a pre-grafting treatment with 6 Gy total body irradiation and i.v. injection of anti-asialo GM1 antibody. One day later, cultured LS 174T human colon cancer cells were injected into the surgically exposed spleen, which was resected 3 min later. In 48 of 65 mice, a few to several hundred liver metastases were macroscopically observed at dissection 3 to 4 weeks after transplantation. Ten of 10 mice, followed-up for survival, died with multiple large confluent liver metastases. By reducing the radiation dose to 4 or 0 Gy, or omitting the anti-asialo GM1 antibody injection, only 60%, 37% or 50% of mice, respectively, had visible metastases 3 weeks after transplantation. Carcinoembryonic antigen (CEA) measured in tumour extracts was in the mean 25.6 micrograms/g in liver metastases compared with 9.2 micrograms/g in s.c. tumours. Uptake of radiolabelled anti-CEA monoclonal antibody (MAb) in the metastases 12, 24 and 48 hr after injection gave a mean value of 39% of the injected dose per gram of tissue (ID/g). In comparison, MAb uptake in s.c. and intrasplenic tumours or lung metastases gave a mean percentage ID/g of 20, 18 and 15, respectively. Laser-induced fluorescence after injection of indocyanin-MAb conjugate allowed direct visual detection of small liver metastases, including some that were not visible under normal light. Preliminary results showed that mice, pre-treated with 4 Gy irradiation and the anti-asialo GM1 injection, were tolerant to radioimmunotherapy with a total dose of 500 muCi 131I labeled anti-CEA intact MAbs given in 3 injections.

Topics: Animals; Antibodies; Antibodies, Monoclonal; Carbocyanines; Carcinoembryonic Antigen; Colonic Neoplasms; Fluorescence; Fluorescent Dyes; G(M1) Ganglioside; Humans; Immunohistochemistry; Lasers; Liver Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Radioimmunotherapy; Tumor Cells, Cultured; Whole-Body Irradiation

Topics: Animals; Benzimidazoles; Biological Transport; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Cells, Cultured; Colonic Neoplasms; Electrophysiology; Fluorescent Dyes; Humans; Intracellular Membranes; Kinetics; Membrane Potentials; Mitochondria; Mitochondria, Heart; Mitochondria, Liver; Oxygen Consumption; Rats; Rats, Sprague-Dawley; Regression Analysis; Spectrometry, Fluorescence; Tumor Cells, Cultured; Uncoupling Agents

The search for improved photosensitizers for laser phototherapy of malignancies has led to the examination of a new group of carbocyanine dyes as effective fluorochromes. In this study, four carbocyanine dyes with different absorption maxima of 483 nm [DiOC6(3)], 545.5 nm (DiIC5(3)], 556.6 nm [DiSC5(3)], and 651.0 nm [DiSC3(5)] were tested in vitro. The kinetics of uptake and toxicity of these four dyes were assessed for P3 human squamous cell carcinoma, HT29 colon carcinoma, M26 melanoma, and TE671 fibrosarcoma cell lines at 15, 30, 45, 60, and 180 minutes after exposure with each dye. After sensitization with DiOC6(3), the P3 and M26 cell lines were also tested for phototherapy by treatment with 488-nm light from an argon laser. The results showed that these four carbocyanine dyes had rapid and significant uptake by the carcinoma cell lines with no toxicity at concentrations < 0.1 micrograms/mL. Nontoxic DiOC6(3) levels in sensitized tumor cells after laser phototherapy resulted in approximately 85% inhibition of P3 and approximately 95% inhibition of M26 cell lines by MTT assays. The results suggest that these carbocyanine dyes can be used for tumor photosensitization and wavelength-matched laser photodynamic therapy. Further in vivo studies will be necessary to define the clinical potential of carbocyanine dyes as tumor-targeting agents for phototherapy of cancer.

Topics: Adenocarcinoma; Argon; Benzothiazoles; Carbocyanines; Carcinoma, Squamous Cell; Cell Survival; Colonic Neoplasms; Fibrosarcoma; Fluorescent Dyes; Humans; Laser Therapy; Lung Neoplasms; Medulloblastoma; Melanoma; Neoplasms; Photochemotherapy; Tetrazolium Salts; Tumor Cells, Cultured

Topics: Antineoplastic Agents; Benzothiazoles; Carbocyanines; Cell Line; Colonic Neoplasms; Fluorescent Dyes; Humans; Membrane Potentials; Mitomycin; Mitomycins

The lateral diffusion of lectin-labelled glycoconjugates was studied in the human colon carcinoma cell line HT29 using fluorescence photobleaching techniques. HT29 cells were grown in either Dulbecco's modified Eagle's medium with glucose (25 mM; DMEM-Glu) or with galactose (25 mM; DMEM-Gal). Cell cultivation in the DMEM-Gal medium was assumed to promote a transformation of the cells to become small-intestinal-like with characteristic microvilli and associated enzymes. The diffusion of glycoconjugates labelled with fluoresceinated Triticum vulgaris agglutinin (Wheat germ agglutinin; WGA), Ricinus communis agglutinin-I (RCA-I), Concanavalia ensiformis agglutinin (ConA), Ulex europaeus agglutinin-I (UEA-I) and Arachis hypogaea agglutinin (PNA) was in all cases rapid, with a diffusion constant (D) ranging between 0.4 and 0.8 X 10(-8) cm2 s-1. As a comparison the diffusion of the fluorescent synthetic lipid analog diI-C14 was characterized by D = 0.8 - 1.0 X 10(-8) cm2 s-1. The diffusion of lectin-labelled surface components could not be related to the presence of microvilli on HT29 cells grown in DMEM-Gal, which ought to yield an apparently lower diffusion rate. The results indicate either that surface glycoconjugates in HT29 cells are dominated by glycolipid, or that the labelled glycoproteins are more or less free to diffuse in the plane of the membrane.

Topics: Adenocarcinoma; Carbocyanines; Cell Line; Colonic Neoplasms; Diffusion; Glycoconjugates; Humans; Kinetics; Lectins; Quinolines; Spectrometry, Fluorescence; Structure-Activity Relationship