Distribution of colon cancer cells permanently labeled by lectin-mediated endocytosis of a trap label.
A method was elaborated for high-yield 125I-trap labeling of rat colon carcinoma cells using conjugates of dichlorotriazine aminofluorescein and bovine serum albumin substituted with either N-acetylgalactosamine or N-acetylglucosamine as vehicles. Fluorescence microscopy revealed that the ligands accumulated in perinuclear vesicles that were probably lysosomes. Monensin inhibited accumulation by 40%, signifying receptor-mediated endocytosis. Competition experiments revealed that the same receptor(s) mediated endocytosis of the two neoglycoproteins. Accumulation of label was greatly enhanced in the absence of serum, resulting in a labeling efficiency of at least 15 cpm/cell, with no sign of toxic effects. At least 75% of the initially accumulated radioactivity resided in the cells 4 days after labeling. After that the loss of radioactivity was linear with time and stabilized at 1.1%/day for at least 2 weeks. Injection of labeled carcinoma cells i.v. into syngeneic rats revealed a very rapid clearance from the circulation. Isolation of the liver cells 24 h later revealed that a great proportion of the administered cells or their remnants had been engulfed by sinusoidal Kupffer and endothelial cells; the parenchymal cells contained a smaller proportion of label. In conclusion, we have developed a technique of labeling colon carcinoma cells with 125I and fluorescein utilizing specific lectin-like receptors for endocytosis. Since the label is trapped intralysosomally, it will also label Kupffer cells and other members of the reticuloendothelial system after internalization. These features make the procedure well suited for studies on the fate of the colon carcinoma cells after administration in vivo. Since the label is trapped intralysosomally for an extended length of time, parameters such as the formation of metastasis and elimination by phagocytosis can readily be determined.
Rushfeldt
C
C
Department of Experimental Pathology and Anatomy, University of Tromsø, Norway.
Smedsrød
B
B
D000116:Acetylgalactosamine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D000230:Adenocarcinoma / Q000097:blood / Q000378:metabolism / Q000473:pathology*; D000818:Animals; D003110:Colonic Neoplasms / Q000097:blood / Q000378:metabolism / Q000473:pathology*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D016650:Fluorescein-5-isothiocyanate / Q000493:pharmacokinetics* / Q000494:pharmacology; D005452:Fluoresceins / Q000493:pharmacokinetics* / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008099:Liver / Q000166:cytology; D008297:Male; D008856:Microscopy, Fluorescence; D008985:Monensin / Q000494:pharmacology; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D051381:Rats; D011919:Rats, Inbred Strains; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000493:pharmacokinetics* / Q000494:pharmacology
D005452:Fluoresceins; D007457:Iodine Radioisotopes; C079431:N-acetylgalactosamine-bovine serum albumin conjugate; C050080:N-acetylglucosamine-bovine serum albumin conjugate; C039083:5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)fluorescein; D012710:Serum Albumin, Bovine; D008985:Monensin; D016650:Fluorescein-5-isothiocyanate; D000116:Acetylgalactosamine; D000117:Acetylglucosamine