Bacillus-Calmette-Guerin (BCG) and 3D tumors: An in vitro model for the study of adhesion and invasion

Purpose: To study adhesion, penetration and internalization of BCC and effe ctor-cells to and into three-dimensional in vitro cell aggregates from beni gn and malignant urothelial origin mimicking small in vitro tumors. Materials and Methods: Multicellular spheroids (MCS) were generated by "liq uid-overlay" technique. Adhesion and penetration of viable FITC-labelled BC G; into MCS from urothelial cancer cell lines and normal urothelial cells w as studied by electron microscopy (TEM) and fluorescence microscopy. Sphero id growth during BCG-co-incubation was determined by light microscopy. Peri pheral blood mononuclear cells (PBMC) were stimulated with BCG to generate BCG-activated-killer (BAK) cells. The infiltration of these effecters and o f lymphokine-activated killer (LAK) cells into MCS was examined at differen t intervals by means of immunohistochemistry. The resulting cytotoxicity wa s judged in a H-3-1-methionine release assay. Results: BCG adhered to MCS from tumor cells but not to benign cell MCS. In tracellular internalization of the bacteria was detectable in superficial t umor cell-layers (1-5) wheras BCG was not found in deeper layers. Prolifera tion of malignant MCS was reduced in the presence of BCG. Benign MCS showed contact inhibition growth arrest, which was not altered by BCG. BAK and LA K effector cells both infiltrated tumor cell MCS as opposed to unstimulated PBMC. In contrast to LAK cells, BAK cells did not infiltrate into benign c ell MCS and were not cytotoxic towards them. Conclusion: With regard to the clinical situation the selective adhesion an d internalization of BCG to malignant cells might explain why BCG has been rarely found in follow-up biopsies in tumor free patients. More interesting ly, the selective adhesion of BCG to and infiltration of BAK effector cells into malignant cell spheroids suggests a selective mode of action of BCG.