The relationship between tumour geometry and the response of tumour cells to cytotoxic drugs — An in vitro study using EMT6 multicellular spheroids

Abstract

Multicellular spheroids of the EMT6/Ca/VJAC mouse mammary tumour cell line have been used in an investigation of the effect of tumour geometry on the response of tumour cells to 3 cytotoxic drugs, adriamycin (ADM), nitrogen mustard (HN2) and CCNU. In addition to the inherent cellular drug response, factors related to spheroid structure, namely cell-cycle distribution, intercellular contact, drug penetration and microenvironment (pH, oxygen, glucose, etc.) are believed to influence the response of cells within spheroids to cytotoxic drugs. Selective enzymatic dissociation (with bacterial neutral protease) has been used to separate the cells within large (approximately 800 micron in diameter) spheroids into 4 distinct subpopulations. The cells within the subpopulations have been characterized by their DNA content, RNA content, tritiated thymidine labelling index, cell size and clonogenic capacity. It was found that cells at the surface of spheroids are relatively larger and more proliferative than cells towards the centre while their clonogenic capacity is similar. Studies on the responses of EMT6/Ca/VJAC log and plateau-phase monolayer cells have been carried out in parallel and have shown that cycling cells are more sensitive to ADM and HN2 than are non-cycling cells but somewhat less sensitive for the response to CCNU. Since the response patterns of cells from different regions of spheroids to HN2, treated either before disaggregation (intact spheroid) or after disaggregation (isolated spheroid cells), are similar and the surviving fraction increases from the surface towards the centre of the spheroid, cell cycle distribution is thought to be the only factor involved in the cytotoxicity of HN2 towards cells within the spheroids. Although the patterns of response to ADM of cells within intact spheroids and isolated spheroid cells are similar to those for HN2, the initial slope of the curve for intact spheroids is much steeper than that of the isolated spheroid cells. Therefore, in addition to the factor of cell-cycle distribution, drug penetration also appears to be involved in the action of ADM on spheroids, while the factors of intercellular contact and microenvironment appear to be relatively less important. The reverse pattern was found for the response of cells within different regions of spheroids to CCNU, treated as intact spheroids or as isolated spheroid cells (i.e., greater killing of inner compared with outer cells).(ABSTRACT TRUNCATED AT 400 WORDS)

Chemosensitization by misonidazole in CCNU-treated spheroids and tumours

“…Both panels of Figure 1 show cellular radiosensitivity as a function of Hoechst staining intensity; in spheroids, the symmetry of the model allows an easy calculation of the depth from which the cells were recovered (Durand, 1983), while in the tumour system, the data are simply presented as fraction number, where fraction I represented the 10% of the cells which were most intensely stained (thus nearest the functional blood supply, e.g. Chaplin et al, 1985Chaplin et al, ,1986a (Figure 2) indicated that the less well oxygenated cells were more sensitive to this nitrosourea, in agreement with previous results obtained with nitrosoureas in other spheroid systems (Deen et al, 1980;Kwok & Twentyman, 1985). Quite reproducible responses were found in the multiple experiments shown (note that in the case of spheroids, different starting sizes led to slightly different estimates of cell depth among experiments).…”

Cytokinetic factors in drug resistance of Lewis lung carcinoma: comparison of cells freshly isolated from tumours with cells from exponential and plateau-phase cultures

“…Similarly, cell proliferation-dependent uptake and cytotoxicity of melphalan have been described (Blosmanis et al, 1987). We have shown that nitrogen mustard is selectively toxic to exponential-phase LLTC cultures (Figure 4c), as previously reported for EMT6 cells (Twentyman & Bleehen, 1975;Kwok & Twentyman, 1985). In contrast chlorambucil, which probably enters cells passively (Goldenberg & Begleiter, 1980) is equally active against exponential and plateau-phase LLTC (Figure 4d).…”

“…The other intercalating agent studied, doxorubicin, also shows higher activity against exponential than against plateau-phase cultures (Figure 4b), as has been demonstrated with EMT6 (Twentyman & Bleehen, 1975;Kwok & Twentyman, 1985), CHO (Barranco & Novak, 1974), L1210 (Bhuyan et al, 1977) and LoVo (Drewinko et al, 1981) cells. Such cycle selectivity of intercalating drugs appears to reflect the activity of topoisomerase II, which declines in noncycling cells (Sullivan et al, 1986).…”

“…Since Bruce and his associates (reviewed by Valeriote & van Putten, 1975;van Putten, 1974) first demonstrated the importance of proliferative state in relation to drug sensitivity in vivo, most studies have utilized in vitro models of non-cycling cells such as plateau-phase cultures (Barranco & Novak, 1974;Drewinko et al, 1981;Hahn & Little, 1972;Twentyman & Bleehen, 1975) and multicellular spheroids (Sutherland & Durand, 1984;Nederman & Twentyman, 1984;Kwok & Twentyman, 1985). The question remains as to whether these provide adequate models for non-cycling cells in tumours (Valeriote & van Putten, 1975;Twentyman & Bleehen, 1975).…”

“…Important differences in drug sensitivity between cycling and non-cycling cells have been demonstrated with antimetabolites, alkylating agents and DNA intercalating drugs (Barranco & Novak, 1974;Drewinko et al, 1981;Kwok & Twentyman, 1985;Twentyman & Bleehen, 1975). The present study focuses on 4-methyl-5-(N-methyl)carboxamideamsacrine (CI-921), a new 9-anilinoacridine derivative which has recently entered phase I clinical trials (Baguley et 1984).…”