texas-red and Adenocarcinoma

This unit describes the methods that we have been developing for analyzing tumor cell motility in mouse and rat models of breast cancer metastasis. Rodents are commonly used to provide a mammalian system for studying human tumor cells as xenografts in immunocompromised mice, as well as for following the development of tumors from a specific tissue type in transgenic lines. The Basic Protocol describes the standard methods used for generation of mammary tumors and imaging them. Additional protocols for labeling macrophages, blood vessel imaging, and image analysis are also included.

Topics: Adenocarcinoma; Animals; Catheters, Indwelling; Cell Line, Tumor; Cell Movement; Female; Green Fluorescent Proteins; Humans; Macrophages; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, SCID; Mice, Transgenic; Microscopy, Fluorescence, Multiphoton; Neoplasm Transplantation; Rats; Rats, Inbred F344; Transfection; Xanthenes

A novel method was developed to measure the effective permeability of microvessels in three-dimensional tumors. Two unique features characterized our approach: (i) Texas Red (with peak excitation and peak emission wavelengths of 596 and 615 nm, respectively) was used for macromolecular labeling, to minimize the absorption of fluorescence light by hemoglobin in blood. Thus the tumor tissue could be treated approximately as a uniform medium with respect to light absorption. (ii) The light absorption and scattering in tumor tissues were accounted for in relating the fluorescence intensity to the amount of Texas Red-labeled macromolecules extravasated. The vascular permeability of Texas Red-labeled bovine serum albumin in human tumor xenograft LS174T implanted in dorsal skin-fold chamber in severe combined immunodeficient mice was measured using this method. The average permeability-surface area product per unit volume (PS/V, x 10(-4) sec-1) and the average effective permeability (P, x 10(-7) cm/sec) were found to be 1.26 +/- 0.72 and 6.06 +/- 4.30, respectively; the fractional volume of tumor vessels (Vves/V, %) was found to be 9.2 +/- 2.9, and the total surface area of vessels per unit volume (S/V, cm2/cm3) was found to be 239 +/- 82. The errors in the estimation of these parameters are discussed. The method described here is general and can be adapted to study the microvascular permeability of superficial tumors in various organs in patients or animals.

Topics: Adenocarcinoma; Animals; Capillary Permeability; Colonic Neoplasms; Fluorescent Dyes; Mice; Mice, SCID; Microcirculation; Microscopy, Fluorescence; Neoplasm Transplantation; Nephelometry and Turbidimetry; Prostheses and Implants; Serum Albumin, Bovine; Skin; Specific Pathogen-Free Organisms; Transplantation, Heterologous; Videotape Recording; Xanthenes

Adenocarcinoma cells often form intracellular lumens and intercellular cysts. In order to study the structural relationships between these lumens and the apical domain of normal enterocytes, we have applied electron microscopy and confocal microscopy to a cloned cell line derived from the human colon adenocarcinoma cell line LoVo which express a high number of intracellular lumens and intercellular cysts. Microvilli reminiscent of those detected in the brush border of small intestinal cells are formed in the two types of compartments. By immunofluorescence, we found that a 135 kDa membrane glycoprotein characterized by a monoclonal Ab and normally associated with the brush-border of enterocytes is expressed at the surface of the intracellular lumens and intercellular cysts present in the adenocarcinoma cells. Comparison of fluorescence and reflection contrast micrographs obtained by confocal microscopy demonstrate the presence of spherical intracellular lumens in the juxtanuclear region of single cells, and of more complex shaped intercellular cysts located within clusters of cells. The later cells form junctional complexes limiting an apical plasma membrane domain in contact with the intercellular cyst. It is suggested that the intracellular lumens may represent the abortive form of an apical plasma membrane due to the lack of components required to establish epithelial cell contacts. As opposed to conventional fluorescence microscopy, confocal microscopy allows rapid inspection of the tridimensional organization of intracellular lumens and intercellular cysts even when they are located in cell multilayers.

Topics: Adenocarcinoma; Cell Compartmentation; Cell Membrane; Colonic Neoplasms; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Microscopy, Electron; Microscopy, Fluorescence; Microvilli; Tumor Cells, Cultured; Xanthenes