carbocyanines and Colorectal-Neoplasms

The innovation of cancer immunotherapy is improving the prognosis of colorectal cancer (CRC) in clinics. Nevertheless, due to tumor heterogeneity and complex underlying inhibitory mechanisms, the therapeutic response greatly varies among different patients. To optimize the clinical management of CRC patients, it is critical to develop novel approaches for response monitoring and prediction. In the current study, we developed a novel near-infrared fluorescence (NIRF) imaging probe (Cy5.5-ICOS mAb) targeting the inducible T-cell costimulatory receptor (ICOS or CD278) and assessed its capacity for the detection of ICOS

Topics: Carbocyanines; Colorectal Neoplasms; Diagnostic Imaging; Fluorescent Dyes; Humans; Immunotherapy

Conventional colonoscopy with white light illumination detects colonic adenomas based on structural changes alone and is limited by a high miss rate. We aim to demonstrate an integrated imaging strategy that combines wide-field endoscopy and confocal endomicroscopy in real time to visualize molecular expression patterns in vivo to detect premalignant colonic mucosa.. A peptide specific for claudin-1 is labeled with Cy5.5 and administrated intravenously in genetically engineered mice that develop adenomas spontaneously in the distal colon. Wide-field endoscopy is used to identify the presence of nonpolypoid and polypoid adenomas. Anatomic landmarks are used to guide placement of a confocal endomicroscope with side-view optics to visualize claudin-1 expression patterns with subcellular resolution.. Wide-field fluorescence images show peak uptake in colon adenoma at ∼1 hour after systemic peptide administration, and lesion margins are clearly defined. Further examination of the lesion using a confocal endomicroscope shows dysplastic crypts with large size, elongated shape, distorted architecture, and variable dimension compared with normal. The mean fluorescence intensity is significantly higher for dysplasia than normal. Increased claudin-1 expression in dysplasia vs normal is confirmed ex vivo, and the binding pattern is consistent with the in vivo imaging results.. Wide-field endoscopy can visualize molecular expression of claudin-1 in vivo to localize premalignant colonic mucosa, and confocal endomicroscopy can identify subcellular feature to distinguish dysplasia from normal.

Topics: Adenoma; Adenomatous Polyps; Animals; Animals, Genetically Modified; Carbocyanines; Claudin-1; Colonic Polyps; Colonoscopy; Colorectal Neoplasms; Fluorescent Antibody Technique; Fluorescent Dyes; Genes, APC; Immunohistochemistry; Mice; Microscopy, Confocal; Peptides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Colorectal cancer is a common malignant tumour with a low 5-year survival rate. A combination therapy with high selectivity and easy controllability is a pressing need for the effective treatment of such cancer. In this study, an indocyanine green derivative (Cy7)-conjugated lipid with a terminal carboxyl group was synthesized, which could self-assemble with a cerasome-forming lipid (CFL) into nanoparticles (NPs) by encapsulating doxorubicin (DOX) to achieve combined photothermal chemotherapy. The resulting Gly@Cy7-Si-DOX NPs with a surface covalent silicate framework showed excellent morphological stability and colloidal stability. Specifically, the conjugated Cy7 was covalently conjugated in the liposomal bilayer, resulting in high drug loading content, high photostability, and high photothermal conversion efficiency, which enabled the resulting nanoparticles to be an effective platform for photothermal therapy. Meanwhile, the encapsulated DOX leaked only slightly under physiological conditions due to the silicate surface of Gly@Cy7-Si-DOX NPs and exhibited controlled release in a weakly acidic environment or under near-infrared (NIR) light irradiation for chemotherapy. Gly@Cy7-Si-DOX NPs were efficiently taken up by tumour cells. Upon light irradiation, the released DOX entered the nuclei of tumour cells, as observed by confocal microscopy and flow cytometry. In vitro cell experiments indicated that both healthy cells and tumour cells were viable under treatment with only Gly@Cy7-Si-DOX NPs, indicating the encapsulated DOX was stably confined to the NPs, and cells were significantly killed when treated with both Gly@Cy7-Si-DOX NPs and NIR laser irradiation. After i.v. administration, Gly@Cy7-Si-DOX NPs accumulated at the tumour site, as monitored by near-infrared fluorescence imaging. A significant tumour inhibition rate (95.8%) was also achieved in a HT-29 colorectal cancer model when treated with Gly@Cy7-Si-DOX NPs plus irradiation. Therefore, the Gly@Cy7-Si-DOX NPs hold great promise for controllable combined colorectal cancer photothermal chemotherapy.

Topics: Animals; Carbocyanines; Colorectal Neoplasms; Coloring Agents; Doxorubicin; Drug Carriers; Female; HT29 Cells; Humans; Infrared Rays; Liposomes; Mice; Nanoparticles; Optical Imaging; Phototherapy; Tissue Distribution

Patients with ulcerative colitis are at increased risk for colorectal cancer and endoscopic surveillance is mandatory. Matrix metalloproteinases (MMPs)-2 and -9 activities are increased in malignant colonic mucosa. The aim of the study was to evaluate molecular imaging of MMP-2/-9 by fluorescence endoscopy (FE) for early tumor detection.. Colorectal cancer in mice (n = 28) was induced by azoxymethane and dextran sodium sulfate. Twenty-four hours after intravenous injection of a nonpeptidic, Cy5.5-labeled MMP-selective tracer, tumor development was assessed in vivo by white light endoscopy and FE. Topical administration of the tracer was also investigated (after 5 minutes and 24 hours). Colonic tumors were evaluated ex vivo by fluorescence reflectance imaging, immunohistochemistry, Western blot analysis, and zymography.. Imaging of MMP-2/-9 expression by FE achieved a significantly higher contrast of the fluorescence signal in colonic adenomas compared with the adjacent nonmalignant mucosa (P < 0.001). Fluorescence reflectance imaging detected a significantly higher tracer uptake in adenoma compared with healthy mucosa (P < 0.001) and revealed a tumor size-dependent increase of tracer uptake (P < 0.01). Topical tracer administration did not facilitate tumor detection. Immunohistochemistry, Western blot analysis, and zymography indicated higher levels of MMP-2 and -9 in high-grade dysplasia and pT1 tumors ex vivo.. MMP-2/-9 expression was significantly increased in colorectal neoplasia. FE allows direct visualization of a prognostic parameter (here MMP-2/-9) on a molecular level and may improve the characterization of colorectal lesions and the adenoma detection rate in the future.

Topics: Animals; Azoxymethane; Blotting, Western; Carbocyanines; Carcinogens; Colorectal Neoplasms; Dextran Sulfate; Endoscopy, Gastrointestinal; Female; Immunoenzyme Techniques; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Optical Imaging

We previously developed a separation-free ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye. This assay could successfully detect one cancer mutation among 10 wild-type templates. In the current study, the mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5) that was spectrally similar but more fluorescent.

Topics: Carbocyanines; Colorectal Neoplasms; DNA; DNA Ligases; DNA Mutational Analysis; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Genes, ras; HT29 Cells; Humans; Ligase Chain Reaction; Ligases; Point Mutation; Quantum Dots; Sensitivity and Specificity

Fucose is utilized for the modification of different molecules involved in blood group determination, immunological reactions, and signal transduction pathways. We have recently reported that enhanced activity of the fucosyltransferase 3 and/or 6 promoted TGF-ß-mediated epithelial mesenchymal transition and was associated with increased metastatic potential of colorectal cancer (CRC), suggesting that fucose is required by CRC cells. With this in mind, we examined requirement of L-fucose in CRC cells and developed fucose-bound nanoparticles as vehicles for delivery of anticancer drugs specific to CRC.. In this study, we first examined the expression of fucosylated proteins in 50 cases of CRC by immunochistochemical staining with biotinylated Aleuria aurantia lectin (AAL). Then we carried out an L-fucose uptake assay using three CRC cell lines. Finally, we developed fucose-bound nanoparticles as vehicles for the delivery of an anticancer drug, SN38, and examined tumor growth inhibition in mouse xenograft model (n = 6 mice per group). All statistical tests were two-sided.. We found a statistically significant relationship between vascular invasion, clinical stage, and intensity score of AAL staining (P ≤ .02). L-fucose uptake assay revealed that L-fucose incorporation, as well as fucosylated protein release, was high in cells rich in fucosylated proteins. L-fucose-bound liposomes effectively delivered Cy5.5 into CRC cells. The excess of L-fucose decreased the efficiency of Cy5.5 uptake through L-fucose-bound liposomes, suggesting an L-fucose receptor dependency. Intravenously injected, L-fucose-bound liposomes carrying SN38 were successfully delivered to CRC cells, mediating efficient tumor growth inhibition (relative tumor growth ratio: no treatment group [NT], 8.29 ± 3.09; SN38-treated group [SN38], 3.53 ± 1.47; liposome-carrying, SN38-treated group [F0], 3.1 ± 1.39; L-fucose-bound, liposome-carrying, SN38-treated group [F50], 0.94 ± 0.89; F50 vs NT, P = .003; F50 vs SN38, P = .02, F50 vs F0, P = .04), as well as prolonging survival of mouse xenograft models (log-rank test, P < .001).. Thus, fucose-bound liposomes carrying anticancer drugs provide a new strategy for the treatment of CRC patients.

Topics: Animals; Antineoplastic Agents, Phytogenic; Camptothecin; Carbocyanines; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Drug Carriers; Drug Delivery Systems; Female; Fucose; Humans; Immunohistochemistry; Irinotecan; Liposomes; Male; Mannose; Mice; Middle Aged; Nanoparticles; Neoplasm Transplantation; Proteins

Somatic mutations in stool DNA are quite specific to colorectal cancer (CRC), but a method being able to detect the extraordinarily low amounts of mutants is challengeable in sensitivity. We proposed a hydrogel bead-array to digitally count CRC-specific mutants in stool at a low cost. At first, multiplex amplification of targets containing multiple mutation loci of interest is carried out by a target enriched multiplex PCR (Tem-PCR), yielding the templates qualified for emulsion PCR (emPCR). Then, after immobilizing the beads from emPCR on a glass surface, the incorporation of Cy3-dUTP into the mutant-specific probes, which are specifically hybridized with the amplified beads from emPCR, is used to color the beads coated with mutants. As all amplified beads are hybridized with the Cy5-labeled universal probe, a mutation rate is readily obtained by digitally counting the beads with different colors (yellow and red). A high specificity of the method is achieved by removing the mismatched probes in a bead-array with electrophoresis. The approach has been used to simultaneously detect 8 mutation loci within the APC, TP53, and KRAS genes in stools from eight CRC patients, and 50% of CRC patients were positively diagnosed; therefore, our method can be a potential tool for the noninvasive diagnosis of CRC.

Topics: Carbocyanines; Colorectal Neoplasms; Deoxyuracil Nucleotides; DNA; Feces; Humans; Multiplex Polymerase Chain Reaction; Mutation; Mutation Rate; Polymerase Chain Reaction; Sensitivity and Specificity

Cell death plays a central role in normal physiology and in disease. Common to apoptotic and necrotic cell death is the eventual loss of plasma membrane integrity. We have produced a small organoarsenical compound, 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid, that rapidly accumulates in the cytosol of dying cells coincident with loss of plasma membrane integrity. The compound is retained in the cytosol predominantly by covalent reaction with the 90 kDa heat shock protein (Hsp90), the most abundant molecular chaperone of the eukaryotic cytoplasm. The organoarsenical was tagged with either optical or radioisotope reporting groups to image cell death in cultured cells and in murine tumors ex vivo and in situ. Tumor cell death in mice was noninvasively imaged by SPECT/CT using an (111)In-tagged compound. This versatile compound should enable the imaging of cell death in most experimental settings.

Topics: Animals; Arsenicals; Carbocyanines; Carcinoma, Lewis Lung; Cell Death; Colorectal Neoplasms; HSP90 Heat-Shock Proteins; Humans; Jurkat Cells; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasms; Pentetic Acid; Peptides; Protein Binding; Radioisotopes

To digitally analyze expression levels of multiple genes in one reaction, we proposed a method termed as 'MDHB' (Multiplexed Digital-PCR coupled with Hydrogel Bead-array). The template for bead-based emulsion PCR (emPCR) was prepared by reverse transcription using sequence-tagged primers. The beads recovered from emPCR were immobilized with hydrogel to form a single-bead layer on a chip, and then decoded by gene-specific probe hybridization and Cy3-dUTP based primer extension reaction. The specificity of probe hybridization was improved by using electrophoresis to remove mismatched probes on the bead's surface. The number of positive beads reflects the abundance of expressed genes; the expression levels of target genes were normalized to a housekeeping gene and expressed as the number ratio of green beads to red beads. The discrimination limit of MDHB is 0.1% (i.e., one target molecule from 1000 background molecules), and the sensitivity of the method is below 100 cells when using the β-actin gene as the detection target. We have successfully employed MDHB to detect the relative expression levels of four colorectal cancer (CRC)-related genes (c-myc, COX-2, MMP7, and DPEP1) in 8 tissue samples and 9 stool samples from CRC patients, giving the detection rates of 100% and 77%, respectively. The results suggest that MDHB could be a potential tool for early non-invasive diagnosis of CRC.

Topics: Carbocyanines; Colorectal Neoplasms; Cyclooxygenase 2; Deoxyuracil Nucleotides; Dipeptidases; DNA Primers; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Hydrogels; Matrix Metalloproteinase 7; Polymerase Chain Reaction; Proto-Oncogene Proteins c-myc

DNA methylation based techniques are important tools in both clinical diagnostics and therapeutics. But most of these methods only analyze a few CpG sites in a target region. Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing.. We have developed a novel approach for quantitative analysis of CpG methylation density on the basis of microarray-based hybridization and incorporation of Cy5-dCTP into the Cy3 labeled target DNA by using Taq DNA Polymerase on microarray. The quantification is achieved by measuring Cy5/Cy3 signal ratio which is proportional to methylation density. This methylation-sensitive technique, termed RMEAM (regional methylation elongation assay on microarray), provides several advantages over existing methods used for methylation analysis. It can determine an exact methylation density of the given region, and has potential of high throughput. We demonstrate a use of this method in determining the methylation density of the promoter region of the tumor-related gene MLH1, TERT and MGMT in colorectal carcinoma patients.. This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample. The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

Topics: 5' Untranslated Regions; Adaptor Proteins, Signal Transducing; Base Sequence; Carbocyanines; Colorectal Neoplasms; CpG Islands; DNA; DNA Methylation; DNA Modification Methylases; DNA Repair Enzymes; DNA, Neoplasm; Fluorescent Dyes; Humans; Molecular Sequence Data; MutL Protein Homolog 1; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Taq Polymerase; Telomerase; Tumor Suppressor Proteins

To elucidate the significance of beta-1,4-galactosyltransferase IV (beta-1,4-GT-IV) in the clinical presentation and prognostication of colorectal cancer.. Tissue lysates from paired tumor and nontumor tissues of a colon cancer patient were labeled separately with fluorescent dyes Cy5 and Cy3 for two-dimensional difference in-gel electrophoresis. Subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and immunoblot analyses identified a down-regulated level of beta-1,4-GT-IV in the tumor tissue. In the follow-up study, paired tissue lysates were obtained from 100 colorectal cancer patients with immunoblot analyses done to compare the levels of beta-1,4-GT-IV expression in these patients.. Of 100 colorectal patients studied, 48% had down-regulated expression of beta-1,4-GT-IV in the tumor tissue but 28% of patients exhibited elevated beta-1,4-GT-IV levels. Increased beta-1,4-GT-IV in the tumor tissue was significantly coexistent with raised serum level of CA-199 and the presence of tumor metastasis (P=0.006 and P<0.001, respectively) but was independent of age and gender of patient, tumor site, tumor size, serum level of carcinoembryonic antigen, grade of tumor cell differentiation, and depth of tumor invasion. The results of logistic regression analyses suggested that tumor beta-1,4-GT-IV overexpression and tumor invasion, but not other patient variables such as tumor size and serum levels of carcinoembryonic antigen and CA19-9, were significantly correlated with the occurrence of metastases (P<0.05). In a multivariate regression analysis, the patient group with tumor beta-1,4-GT-IV overexpression strongly predicted for tumor metastasis (odds ratio, 10.009; 95% confidence interval, 2.992-33.484; P<0.001). Likewise, tumor beta-1,4-GT-IV overexpression was significantly associated with poor overall survival (P<0.01). By Cox regression analysis, this association remained significant even after adjustment for tumor metastasis (P=0.048).. Increased beta-1,4-GT-IV expression in tumor tissue was strongly associated with tumor metastases and poor prognosis in colorectal cancer.

Topics: Aged; Carbocyanines; Colorectal Neoplasms; Electrophoresis, Gel, Two-Dimensional; Female; Galactosyltransferases; Humans; Male; Middle Aged; Neoplasm Metastasis; Prognosis; Risk Factors; Up-Regulation