Current therapeutic treatments (surgery, radiation, chemotherapy or a combination thereof) are ineffective in increasing survival of patients with advanced stages of squamous carcinomas. New therapeutic directions are needed and differentiation based therapy has been proposed to be one such avenue. This project is aimed at characterizing and determining the efficacy of a multicellular tumor spheroid (MTS) model as a preclinical tool for studying differentiation therapy of squamous carcinomas. In this model tumor cells grow in three dimensions and MTSs that exhibit both morphological and biochemical evidence for squamous differentiation and MTSs that are undifferentiated will be used. Changes in proliferation (growth studies, DNA synthesis, Ki-67 nuclear antigen expression) and differentiation markers (involucrin, filaggrin, keratin, transglutaminase, cornified envelopes) will be determined using biochemical, immunological and immunohistochemical techniques with MTSs of increasing size (initial dia. 250-300, 550-600, greater than 1000 mm). Subsequently, MTS studies will use differentiation modulating agents which are currently being evaluated in clinical trials and which are also known to either suppress the squamous phenotype (retinoids) or induce it (interferons). Drug induced effects on growth and differentiation will be determined. Additionally, individual cell proliferative potentials will be determined through MTS clonogenity and outgrowth studies, labeling index and flow cytometric analyses. Our hypothesis for the model is that if differentiation were induced, MTS growth should be decreased, differentiation markers increased, and proliferatively, individual cells should exit the cell cycle (increase in G1 population) and differentiate. Parallel drug penetration studies will define the potential diffusion barrier in this tissue-like model and liposomes will be examined as a delivery system to overcome any diffusion problems. In corollary to the MTS model, monolayer cultures will be similarly examined. A newly observed effect of retinoids (i.e. continuing cell proliferation in what appears to be a growth inhibited state, mechanistically related to RA induced cell death) will be examined in more detail to understand the universality of the phenomenon and the kinetics and mechanisms of cell death; the potential of combination therapy with cytotoxic agents will also be investigated to take advantage of the continuing cellular proliferation. With other differentiation agents. combination therapy will also be investigated to determine whether cytotoxicity can be potentiated through modulation of SCC differentiation status. Once established, the system could be used to identify agents with potential therapeutic activity in modulating squamous differentiation and as a system to investigate basic mechanisms associated with drug induced differentiation. Using such modulators of differentiation, these models could provide preclinical evaluation of agents for extension of differentiation-oriented ongoing clinical trials in our institution.
