Doxorubicin uptake in ascitic lymphoma model: Resistance or curability is governed by tumor cell density and prolonged drug retention

Abstract

Background/Aims: Chemotherapy resistance of malignancies is a universal phenomenon which unfavorably affects therapeutic results. Genetic adaptations as well as epigenetic factors can play an important role in the development of multidrug resistance. Cytotoxic drug content in plasma of cancer patients is known to variate up to one hundred-fold regardless of the same dose injected per m2 body surface. The relationship between plasma concentrations, tissue uptake, and chemotherapy response is not completely understood. The main objective of this study was to investigate how the identical dose of Doxorubicin (Dox) can result in a different therapeutic response pattern depending on tumor size. Study Design: The study was performed on ascitic EL4 lymphoma in an exponential growth phase focusing on the rapidly changing tumor susceptibility to the Dox treatment. Well distinguishable tumor response patterns (curability, remission-relapse, resistance) were selected to unveil Dox intratumoral uptake and drug tissue persistence. Intratumoral Dox content within peritoneal cavity (PerC) in conjunction with systemic toxicity and plasma pharmacokinetics, were monitored at several time points following Dox injection in tumor bearing mice (TBM) with differing patterns of response. Results: Following intraperitoneal (i.p.) transplantation of 5x104 EL4 lymphoma cells rapid exponential proliferation with ascites volume and animal mass increase resulted in median survival of 14.5 days. The increase in tumor cell mass in PerC between day 3 and day 9 was 112.5-fold (0.2±0.03 mg vs 22.5±0.31 mg respectively). However, tumors at this time interval (day 3 to day 9 post-transplantation) were relatively small and constituted less than 0.05% of animal weight. An identical dose of Dox (15 mg/kg) injected intravenously (i.v.) on Day 3 lead to a cure whereas a TBM injected on day 9 exhibited resistance with a median survival time no different from the untreated TBM control. Injection of Dox resulted in noticeable differences of cellular uptake in PerC between all three groups of TBM (“cure”, relapse”, “resistance”). Larger tumors were consistently taking up less Dox 60 min after the 15 mg/kg i.v. bolus injection. Higher initial uptake resulted also in longer retention of drug in PerC cells. The area under the concentration curve in PerC cells AUC0-10d was 8.2+0.57 µg/g x h, 4.6+0.27 µg/g x h and 1.6+0.02 µg/g x h in “cure”, “relapse” and “resistance” TBM respectively (p<0.05 “relapse” vs “cure” and p<0.001 “resistance” vs “cure”). No differences in plasma Dox pharmacokinetics or systemic hematological effects were observed in TBM following a single i.v. Dox push. Hematologic nadir was tested on day 2 and subsequent hematologic recovery was evaluated on day 10 following Dox administration. Hematologic recovery on day 10 coincided with complete drug efflux from PerC and rising tumor cell numbers in PerC of “relapse” TBM. Myelosuppression and hematological recovery patterns were identical in all surviving animal groups regardless of the tumor size on the day of Dox injection. Conclusions: Within a few days of exponential tumor growth, an identical dose of Dox produced dramatically different responses in the TBM with increasing resistance. Systemic toxicity and plasma pharmacokinetics were indistinguishable between all TBM groups. Initial uptake in tumor cells was found to be consistently lower in larger tumors. Drug uptake in tumor cells was regulated locally - a phenomenon known as inoculum effect in vitro. The duration of drug retention in cells was directly related to initial cellular uptake. The magnitude of Dox cellular retention could potentially play a role in determining tumor remission and relapse.