INTRACELLULAR DOXORUBICIN KINETICS IN LYMPHOMA-CELLS AND LYMPHOCYTES INFILTRATING THE TUMOR AREA IN-VIVO - A FLOW CYTOMETRIC STUDY

Recently we have reported the development of a safe and effective chem immunotherapy protocol involving doxorubicin (Dox) in combination with interleukin 2 which, in C57BL/6 mice, boosts local T cell responses, and, in 50 to 80% of the cases, this resulted in the complete eradicat ion of established syngeneic EL4 lymphoma or its Dox-resistant variant , EL4/A. Accumulation of host-derived leukocytes in the peritoneal cav ity was increased up to 8-fold after tumor inoculation, but, in absolu te numbers, did not increase further following Dox administration. The cellular pharmacokinetic studies undertaken to clarify the role of Do x following a single IV injection indicate that 4 h later, lymphocytes found in the peritoneal cavity have detectable levels of Dox; but the lymphoma cells (both EL4 and EL4/A) have, in proportion to their larg er size, taken up more drug as judged by flow cytometry. The estimated drug ''concentration'' (i.e., intracellular amount divided by estimat ed cell size) at the 4-h time point, however, was found to be essentia lly equivalent in both the lymphoma cells and the lymphocytes. Thereaf ter, the drug content and intracellular ''concentration'' in the EI4/A cells rapidly declined while their numbers progressively increased. I n contrast, the EL4 lymphoma cells and the lymphocytes found in the pe ritoneal cavity in the presence of either lymphoma consistently exhibi ted higher levels of drug at 24-48 h than at 4 h. Splenic and tumor-in filtrating mature T (CD3(+)) cells were completely insensitive to Dox cytotoxicity and actually showed increased CTL activity when examined ex vivo. Although EL4 cells had identical Dox uptake patterns to those of CD3(+) cells, they were sensitive to the drug and their numbers de creased, resulting in increased host/tumor cell ratios in these mice. The pharmacokinetic parameters of the drug and the insensitivity of th e mature T cells to the drug determined in this study can explain, in part, the efficacy of a chemoimmunotherapy protocol boosting local T-c ell responses.