The relationship between tumour geometry and the response of tumour cells to cytotoxic drugs — Anin 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 ( ≈ 800 μm in diameter) spheroids into 4 distinct subpopulations. The cells within the subpopulations have been characterized by their DNA content, RNA content, tritiated thymidine labelling in dex, 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 response 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 penetraction 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). There was, however, a much greater differential effect when treatment was carried out on intact spheroids compared with isolated spheroid cells. As ¹⁴C‐CCNU is found to be distributed consistently through out the whole spheroid, both cell‐cycle distribution and changes in the cellular microenvironment within the spheroids appear to play important roles in the response of cells within spheroids to CCNU.