Communication between Malignant Cells Dieter F. Hülser Biophysics Department, Biology Institute, University of Stuttgart, Germany

Monolayer of embryonic chicken heart cells Synchronous beating by signal transfer through gap junctions Are monolayers representative for the function of an organ or tissue? Intercellular Communication

Communication Hypothesis Tumour cells can be discriminated from normal cells by their lack of communication 1970: J. Sheridan as well as L. Boitsova et al. Same and other tumours were found to be coupled. 1967: W. Loewenstein & Y. Kanno Liver cells are electrically coupled, hepatoma cells are not.

Modified Communication Hypothesis Temporary reduction of coupling during malignant transformation Cannot be tested under in vivo conditions

Seven neoplastic cell lines were derived from BD IX fetal brain cells in culture. All were gap junctionally coupled. Three neoplastic cell lines originating from neuroectodermal BD IX rat tumours were coupled. Uncoupled clones were selected from two of these established cell lines after longer cultivation periods.

Gap Junction BICR/M1R k cells, EM thin section, lanthanum staining

M.-L. Rütz & D. F. Hülser 2000 Crystalline Gap Junction Plaque 7 BICR/M1R k cells, freeze fractured

Human Connexins α β γ unknown

Human Connexins α β γ non grouped

Loss of Growth Control, but No Loss of Connexin Astrocytomas and oligodendrogliomas express the same Cx43 and Cx32 as their normal origin. The abundance of connexins is not correlated with the severity of the tumours. The escape from growth control does not appear to result from the absence of gap junctional communication.

Gap Junction Coupling BICR/M1R k Rat Mammary Carcinoma Cells Lucifer yellow injection De-adaptation during long term cultivation Cx26 and Cx32 would be characteristic for glands, instead only Cx43 is expressed

Regulation of Junctional Coupling 12 BICR/M1R cells, 2 days old multicell spheroid Calcein transfer

BICR/M1R cells, 4 days old multicell spheroid, reduced calcein transfer 13 Regulation of Junctional Coupling

Radioresistance γ-irradiation coupled cells and 3-dimensionally grown cells have the highest survival rates

Metabolic cooperation Cell survival Cell killing Coordinated responses and synchronized actions Basis for therapeutic strategy Bystander Hypothesis

HeLa cells, originating from a human cervix carcinoma, exist as coupled as well as uncoupled clones. Connexin-deficient HeLa cells can be transfected with selected connexin genes. These clones allow functional analyses of distinct gap junction channels and reveal some regulatory properties. Test in a confrontation-invasion assay Test of Bystander Hypothesis

Confrontation Embryonic chicken brain and heart Both have Cx43 gap junction channels Different homotypic cell adhesion No invasion

18 Confrontation HeLa - embryonic chicken heart HeLa have no gap junction channels Homo- or heterotypic cell adhesion? No invasion

Confrontation C6-glioma - embryonic chicken heart Both have Cx43 gap junction channels Heterotypic cell adhesion Invasion

20 Confrontation HeLaCx43 - embryonic chicken heart Both have Cx43 gap junction channels Heterotypic cell adhesion Invasion

Summary Normal and malignant cells express gap junction channels. Tumour cells may have non-typical connexin types. Non-coupled cells can be obtained by culture conditions. Gap junction channels maintain a bystander effect. Coupled cells are less sensitive to irradiation. Gap junction channels facilitate invasion of tumour cells. Regulatory processes may not be detected in cell pairs and in monolayer cells, but can be effective in multicell-spheroids and in vivo.