sepharose and Adenocarcinoma

Intraoperative molecular imaging (IMI) with folate-targeted NIR tracers has been shown to improve lesion localization in more than 80% of lung adenocarcinomas. However, mucinous adenocarcinomas (MAs) and invasive mucinous adenocarcinomas (IMAs) of the lung, which are variants of adenocarcinoma, appear to have decreased fluorescence despite appropriate folate receptor expression on the tumor surface. We hypothesized that the etiology may be related to light excitation and emission through non-Newtonian fluid (mucin) produced by goblet and columnar cancer cells.. Intraoperative data for 311 subjects were retrospectively reviewed from a prospectively collected 6-year database. For standardization, all patients underwent infusion of the same targeted molecular optical contrast agent (pafolacianine, folate receptor-targeted NIR fluorochrome) for lung cancer resections. Then, the ratio of the mean fluorescence intensity of the tumors and background tissues (TBR) was calculated. Tumors were examined for mucin, FRa, FRb, and immunofluorescent tracer uptake by a board-certified pathologist. The optical properties of mucin analyzed by imaging software were used to create in vitro gel models to explore the effects on NIR tracer fluorescence intensity.. A large proportion (192, 62%) of the patients were female, with an average of 62.8 years and a 34-year mean pack smoking history. There were no severe (Clavien-Dindo > III) complications related to pafolacianine infusion. A total of 195 lesions in the study were adenocarcinomas, of which 19 (6.1%) were of the mucinous subtype. A total of 14/19 of the patients had a smoking history, and more than 74% of the IMA lesions were in the lower lobes. IMA lesions had a lower in situ TBR than nonmucinous adenocarcinomas (2.64 SD 0.23) vs (3.45 SD 0.11), respectively (p < 0.05). Only 9/19 (47%) were localized in situ. Tumor bisection and removal of mucin from IMAs significantly increased pafolacianine fluorescence, with resultant TBR not being significantly different from the control group (4.67 vs 4.89) (p = 0.19). Of the 16 lesions that underwent FR expression analysis, 15/16 had FR presence on cancer cells or tumor-associated macrophages in the tumor microenvironment. There was no statistically significant difference in fluorescence intensity during immunofluorescence analysis (4.99 vs 5.08) (p = 0.16). Physical removal of mucin from IMAs improved the TBR from 3.11 to 4.67 (p < 0.05). In vitro analysis of the impact of synthetic non-Newtonian fluid (agarose 0.5%) on NIR tracer fluorescence showed a decrease in MFI by a factor of 0.25 regardless of the concentration for each 5 mm thickness of mucin.. The mucinous subtype of lung adenocarcinomas presents a unique challenge in pafolacianine-targeted IMI-guided resections. The presence of non-Newtonian fluids presents a physical barrier that dampens the excitation of the tracer and fluorescence emission detected by the camera. Knowledge of this phenomenon can allow the surgeon to critically analyze lesion fluorescence parameters during IMI-guided lung cancer resections.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Adenocarcinoma, Mucinous; Contrast Media; Female; Fluorescent Dyes; Folic Acid; Humans; Lung; Lung Neoplasms; Male; Middle Aged; Molecular Imaging; Mucins; Retrospective Studies; Sepharose; Tumor Microenvironment

Innovative diagnostic methods are the need of the hour that could complement conventional histopathology for cancer diagnosis. In this perspective, we propose a new concept based on spectral histopathology, using IR spectral micro-imaging, directly applied to paraffinized colon tissue array stabilized in an agarose matrix without any chemical pre-treatment. In order to correct spectral interferences from paraffin and agarose, a mathematical procedure is implemented. The corrected spectral images are then processed by a multivariate clustering method to automatically recover, on the basis of their intrinsic molecular composition, the main histological classes of the normal and the tumoral colon tissue. The spectral signatures from different histological classes of the colonic tissues are analyzed using statistical methods (Kruskal-Wallis test and principal component analysis) to identify the most discriminant IR features. These features allow characterizing some of the biomolecular alterations associated with malignancy. Thus, via a single analysis, in a label-free and nondestructive manner, main changes associated with nucleotide, carbohydrates, and collagen features can be identified simultaneously between the compared normal and the cancerous tissues. The present study demonstrates the potential of IR spectral imaging as a complementary modern tool, to conventional histopathology, for an objective cancer diagnosis directly from paraffin-embedded tissue arrays.

Topics: Adenocarcinoma; Algorithms; Carbohydrates; Collagen; Colonic Neoplasms; Diagnostic Imaging; Humans; Nucleotides; Optical Phenomena; Paraffin Embedding; Principal Component Analysis; Sepharose; Spectrophotometry, Infrared; Spectroscopy, Fourier Transform Infrared; Tissue Array Analysis

The evaluation of new drug treatments and combination treatments for gliomas and other cancers requires a robust means to interrogate wide dose ranges and varying times of drug exposure without stain-inactivation of the cells (colonies). To this end, we developed a 3-dimensional (3D) colony formation assay that makes use of GelCount technology, a new cell colony counter for gels and soft agars. We used U251MG, SNB19, and LNZ308 glioma cell lines and MiaPaCa pancreas adenocarcinoma and SW480 colon adenocarcinoma cell lines. Colonies were grown in a two-tiered agarose that had 0.7% agarose on the bottom and 0.3% agarose on top. We then studied the effects of DFMO, carboplatin, and SAHA over a 3-log dose range and over multiple days of drug exposure. Using GelCount we approximated the area under the curve (AUC) of colony volumes as the sum of colony volumes (microm2xOD) in each plate to calculate IC50 values. Adenocarcinoma colonies were recognized by GelCount scanning at 3-4 days, while it took 6-7 days to detect glioma colonies. The growth rate of MiaPaCa and SW480 cells was rapid, with 100 colonies counted in 5-6 days; glioma cells grew more slowly, with 100 colonies counted in 9-10 days. Reliable log dose versus AUC curves were observed for all drugs studied. In conclusion, the GelCount method that we describe is more quantitative than traditional colony assays and allows precise study of drug effects with respect to both dose and time of exposure using fewer culture plates.

Topics: Adenocarcinoma; Agar; Antineoplastic Agents; Area Under Curve; Carboplatin; Cell Line, Tumor; Cell Proliferation; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Inhibitory Concentration 50; Sepharose

Glycosaminoglycans (GAGs) in proteoglycan (PG) forms or as free GAGs are implicated in the growth and progression of malignant tumors. These macromolecules were investigated in human gastric carcinoma (HGC) and compared with those in human normal gastric mucosa (HNG). We report that HGC contained about 2-fold increased amounts of GAGs in comparison to HNG. Specifically, HGC showed 3- and 2.5-fold net increase in chondroitin sulphate (CS) and hyaluronan (HA) contents, respectively. Dermatan sulphate (DS) was slightly increased, but the amount of heparan sulphate (HS) was decreased. Of particular, interest were the quite different sulphation profiles of CS and DS chains in HGC in which, non-sulphated and 6-sulphated disaccharide units were increased 10 and 4 times, respectively, in comparison to HNG. On PG level, three different populations were identified in both HNG and HGC, being HSPGs, versican (CS/DS chains) and decorin (CS/DS chains). In HGC, the amounts of versican and decorin were significantly increased about 3- and 8-fold, respectively. These PGs were also characterized by marked decrease in hydrodynamic size and GAG content per PG molecule. Analysis of Delta-disaccharide of versican and decorin from HGC showed an increase of 6-sulphated Delta-disaccharides (Delta di-6S) and non-sulphated Delta-disaccharides (Delta di-0S) with a parallel decrease of 4-sulphated Delta-disaccharides (Delta di-4S) as compared to HNG, which closely correlated with the increase of CS content. In addition, the accumulation of core proteins of versican and decorin in HGC was also associated with many post-translational modifications, referring to the number, size, degree and patterns of sulphation and epimerization of CS/DS chains. Studies on the modified metabolism of PGs/GAGs are under progress and will help in deeper understanding of the environment in which tumor cells proliferate and invade.

Topics: Adenocarcinoma; Cell Division; Chondroitin Sulfate Proteoglycans; Decorin; Extracellular Matrix Proteins; Female; Gastric Mucosa; Glycosaminoglycans; Humans; Lectins, C-Type; Male; Middle Aged; Proteoglycans; Sepharose; Stomach Neoplasms; Subcellular Fractions; Versicans

Human colon cancer cell lines express epidermal growth factor (EGF) mRNA, secrete EGF and may respond to it via the cell-surface EGF receptor (EGFR). Expression of these molecules in human colon and colon tumor, however, is not clear. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of RNA prepared from paired normal human colon and colon tumor samples from 12 individuals followed by Southern blotting analyses of the RT-PCR products revealed a major fragment of 527 bp and a minor fragment of 404 bp that hybridized to a human EGF cDNA probe under stringent conditions. Identical results were obtained from 8 human colon cancer cell lines. Cloning and sequencing of PCR products confirmed that both fragments were from the human EGF gene; the 527-bp fragment corresponded exactly to nucleotides 2,891 to 3,417 of the human EGF mRNA reported by others. A deletion of 123 nucleotides (nucleotides 3,172 to 3,294) was found in the 404-bp fragment. Immunohistochemical studies using cyostat sections of human colon specimens showed that EGF was expressed in the human colon and that expression was restricted to the epithelial colonic crypt cells and epithelium-derived cancer cells. Since EGF and EGF-related molecules are potent mitogens that mediated their effect through the EGFR, we also determined the efficacy of anti-sense EGFR RNA in circumventing the EGFR-related pathway of proliferation. Expression of anti-sense EGFR RNA, by transfection with an inducible anti-sense EGFR expression vector, down-regulated cell-surface EGFR expression and proliferation of these cells and their ability to grow in soft agar. Anti-sense EGFR RNA was found to be an anti-proliferative agent in both relatively non-aggressive and highly aggressive human colon cancer cells.

Topics: Adenocarcinoma; Base Sequence; Blotting, Southern; Cell Division; Cloning, Molecular; Colon; Colonic Neoplasms; Down-Regulation; Epidermal Growth Factor; Epithelium; ErbB Receptors; Humans; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Antisense; RNA, Messenger; Sepharose; Transcription, Genetic; Transfection; Tumor Cells, Cultured

A gel electrophoretic method coupled with agar diffusion has been devised for detecting tumor antigens in human colon tissue. Separation of the antigens is achieved on duplicate electrophoretic gels. One gel is used for the location of the antigens by protein staining and the other gel is used for assaying of the antigenicity by agar diffusion against homologous antiserum. Analysis of perchloric acid extracts of colon tumors by this coupled method revealed the presence of carcinoembryonic antigen and two additional glycoprotein antigens. Analysis of KCl-HCl tumor extracts revealed two new tumor antigens.

Topics: Adenocarcinoma; Antigens, Neoplasm; Carcinoembryonic Antigen; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Humans; Immunodiffusion; Perchlorates; Sepharose

We combined a vector decomposition technique with Gaussian probability thresholding in feature space to segment normal brain tissues, tumors, or other abnormalities on dual-echo MR images. The vector decomposition technique assigns to each voxel a fractional volume for each of two tissues. A probability threshold, based on an assumed Gaussian probability density function describing random noise, isolates a region in feature space for fractional volume calculation that minimizes contamination from other tissues. The calculated fractional volumes are unbiased estimates of the true fractional volumes. The contrast-to-noise ratio (CNR) between tissues on the segmented images is the same as the Euclidean norm of CNRs in the original images. The method is capable of segmenting more than two tissues from a set of dual-echo images by sequentially analyzing different pairs of tissues. The model is analyzed mathematically and in experiments with a phantom. Two clinical examples are presented.

Topics: Adenocarcinoma; Algorithms; Artifacts; Brain; Brain Neoplasms; Cerebrospinal Fluid; Copper; Copper Sulfate; Cysts; Gels; Humans; Image Enhancement; Image Processing, Computer-Assisted; Magnetic Resonance Imaging; Models, Biological; Models, Structural; Models, Theoretical; Multiple Sclerosis; Probability; Sepharose

This paper describes the evaluation of a colony formation assay using automated image analysis, which permits the tracking of growth at the individual colony level, such that a growth rate can be estimated for each colony followed. In principle, this will permit quantitative characterization of cellular heterogeneity in growth rate and cellular heterogeneity in response to proliferation-modifying agents. In addition, we have demonstrated the possibility of using correlative microscopy to relate growth rate to other parameters, using metabolic viability as an example. This should be useful for determining cellular characteristics associated with proliferative behavior and response to proliferation-modifying agents.

Topics: Adenocarcinoma; Cell Transformation, Neoplastic; Humans; Ileal Neoplasms; Image Processing, Computer-Assisted; Sepharose; Tumor Cells, Cultured

We tested the ability of serum-free media to support the in vitro growth of human non-small cell lung carcinoma. A medium containing insulin, transferrin, sodium selenite, hydrocortisone, epidermal growth factor, and bovine serum albumin (1 mg/ml) with serum precoating of culture dishes (modified LA medium) supported three previously established cell lines of non-small cell lung cancer and prevented fibroblast proliferation in fresh tumor specimens but did not support long term tumor cell growth from fresh specimens. We added triiodothyronine, sodium pyruvate, and additional glutamine, insulin, and epidermal growth factor to modified LA medium, precoated with fibronectin and collagen instead of serum, and deleted bovine serum albumin, defining a new medium called ACL-3. ACL-3 medium alone supported the short term growth of 10 of 12 cell lines and the soft agarose cloning of 9 of 12 cell lines tested, and ACL-3 supplemented by an optimal concentration of bovine serum albumin (5 mg/ml) supported the long term growth of 10 of 12 cell lines tested. Moreover, we have grown tumor cells for more than 6 months from 11 of 33 (33%) consecutive fresh clinical specimens of human lung adenocarcinoma in ACL-3 with bovine serum albumin. ACL-3 medium provides a defined environment for the study of growth factor requirements of human non-small cell lung cancer and enhances our ability to grow human lung cancer, particularly adenocarcinoma, in vitro.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Cell Division; Cell Line; Culture Media; Culture Techniques; Extracellular Matrix; Growth Substances; Humans; Lung Neoplasms; Sepharose

A soft agarose clonogenic assay is presented which has been optimized for the growth of human gastrointestinal adenocarcinomas. Samples from 15 gastric and colonic solid tumors and from 2 noncancerous stomachs (control cultures) were disaggregated by treatment with collagenase at 37 degrees overnight. Colonies appeared 10 to 15 days after plating, with a cloning efficiency between 0 and 0.82%, which was markedly improved by a fibroblastic feeder layer. The results suggest a correlation between cloning efficiency and the degree of differentiation of the initial tumor. Histochemistry, electron microscopy, and a carcinoembryonic antigen immunofluorescence assay showed that the colonies consisted of cells with the same characteristics as those of the original tumor.. This colony formation assay appears to be potentially useful for assessing the stem cell pool of gastrointestinal tumors. It will be valuable for studying their response to chemotherapeutic agents in vitro. This clonogenic assay may also permit the establishment of cancer cell lines.

Topics: Adenocarcinoma; Carcinoembryonic Antigen; Cell Differentiation; Cell Division; Cell Line; Cells, Cultured; Colonic Neoplasms; Colony-Forming Units Assay; Gastrointestinal Neoplasms; Humans; Sepharose; Stomach Neoplasms

Topics: Adenocarcinoma; Animals; Cell Division; Cells, Cultured; Culture Media; Female; Karyotyping; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Neoplasm Transplantation; Neoplasms, Experimental; Sepharose

We are isoelectric focusing small amounts of tissues placed directly on electroendosmosis-free agarose gels instead of using the saline extracts of tissue homogenates. More numerous proteins are extracted during isoelectric focusing of the solid tissues than present in the same tissues.

Topics: Adenocarcinoma; Colonic Neoplasms; Histological Techniques; Humans; Isoelectric Focusing; Neoplasm Proteins; Sepharose; Time Factors

We describe here the use of a new agarose for isoelectric focusing of body fluids and tissue proteins. Macromolecular proteins even when significantly greater in size than 1 X 10(6) mol.wt. were easily and rapidly focused.

Topics: Adenocarcinoma; Ascitic Fluid; Blood Proteins; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Humans; Isoelectric Focusing; Neoplasm Proteins; Polysaccharides; Sepharose

The agarose microdroplet leucocyte migration technique is an in vitro technique for detection of cell-mediated hypersensitivity in man, using only 10-15% of the volume of blood required for other leucocyte migration techniques. The technique is described in detail and results are presented from migrations with leucocytes from hypernephroma patients and controls tested with hypernephroma extract and normal kidney extract, and from tuberculin-positive and tuberculin-negative persons tested with PPD.

Topics: Adenocarcinoma; Aged; Antigens, Neoplasm; Cell Migration Inhibition; Dose-Response Relationship, Immunologic; Female; Humans; Hypersensitivity, Delayed; Kidney Neoplasms; Leukocytes; Male; Middle Aged; Sepharose; Tuberculin

Topics: Adenocarcinoma; Animals; Binding Sites, Antibody; Carcinoembryonic Antigen; Chromatography, Affinity; Colonic Neoplasms; Goats; Humans; Immunoelectrophoresis; Immunoglobulin G; Iodine Radioisotopes; Liver Neoplasms; Neoplasm Metastasis; Radioimmunoassay; Sepharose

Topics: Adenocarcinoma; Aluminum Hydroxide; Animals; Animals, Newborn; Antibody Formation; Carcinoembryonic Antigen; Colonic Neoplasms; Goats; Horses; Immune Sera; Immune Tolerance; Immunization, Secondary; Immunodiffusion; Immunoelectrophoresis; Liver Neoplasms; Neoplasm Metastasis; Rabbits; Radioimmunoassay; Sepharose