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 effector-cells to and into three-dimensional in vitro cell aggregates from benign and malignant urothelial origin mimicking small in vitro tumors. Materials and Methods: Multicellular spheroids (MCS) were generated by "liquid-overlay" technique. Adhesion and penetration of viable FITC-labelled BCG; into MCS from urothelial cancer cell lines and normal urothelial cells was studied by electron microscopy (TEM) and fluorescence microscopy. Spheroid growth during BCG-co-incubation was determined by light microscopy. Peripheral blood mononuclear cells (PBMC) were stimulated with BCG to generate BCG-activated-killer (BAK) cells. The infiltration of these effecters and of lymphokine-activated killer (LAK) cells into MCS was examined at different intervals by means of immunohistochemistry. The resulting cytotoxicity was judged in a H-3-1-methionine release assay. Results: BCG adhered to MCS from tumor cells but not to benign cell MCS. Intracellular internalization of the bacteria was detectable in superficial tumor cell-layers (1-5) wheras BCG was not found in deeper layers. Proliferation 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 LAK effector cells both infiltrated tumor cell MCS as opposed to unstimulated PBMC. In contrast to LAK cells, BAK cells did not infiltrate into benign cell MCS and were not cytotoxic towards them. Conclusion: With regard to the clinical situation the selective adhesion and internalization of BCG to malignant cells might explain why BCG has been rarely found in follow-up biopsies in tumor free patients. More interestingly, the selective adhesion of BCG to and infiltration of BAK effector cells into malignant cell spheroids suggests a selective mode of action of BCG.