Abstract B49: Cancer cell spheroids as a model to study sequential chemotherapy protocols

This study aimed at comparing six combinations of priming drug / doxorubicin (DOX) in a spheroid model. Three-dimensional cultures of drug-resistant cancer cells (spheroids) are physiologically relevant[1] and have been used as models of limited drug penetration[2]. Enhanced tumor accumulation and therapeutic efficacy of liposomal DOX after apoptosis induction by paclitaxel (PTX) has been reported[3]. Moreover, mitochondrial depolarization is correlated with clinical response[4]. Here we proposed use of spheroids to evaluate sequential chemotherapy combinations for potentiation of DOX cytotoxicity after pretreatment with cytotoxic concentrations of clinically relevant drugs. To this aim, 400–500 μm NCI-ADR-RES (ovarian carcinoma) were used. We first incubated spheroids 48h with mitoxantrone (MXO), cisplatin (CIS), dexamethasone (DXM), methotrexate (MTX), sclareol (SCL) and PTX as first line (priming) agents at concentrations inducing a 1.5- to 2.5-fold lactate dehydrogenase (LDH) release compared to untreated cells. For DOX accumulation studies, these were further cultured in the presence of 25 μM of DOX during 24h before determination of DOX content (nanomoles DOX/mg of proteins) by fluorescence. For DOX cytotoxicity evaluation, pretreated spheroids were incubated 72h with 100 μM of DOX before assessment of viability with a Cytotox 96 nonradioactive cell viability kit (Promega). Three DOX forms were used: free drug (DOX), distearyl1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[amino(polyethylene glycol)-2000] / DOX micelles (MDOX, 13 nm) prepared according to [5, 6], and liposomal DOX (LDOX, Lipo-Dox®, 130 nm, Sun Pharma India). The accumulation pattern of these forms was DOX = MDOX > LDOX as previously reported[7]. We document enhanced DOX, MDOX and LDOX accumulation after spheroids pretreatment with mitoxantrone (2.8-; 1.8- and 2.5-fold, respectively) and increased distribution of DOX and MDOX in PTX primed spheroids (2- and 1.4-fold, respectively). CIS, DXM, SCL and MTX failed at enhancing free, micellar or liposomal DOX distribution in spheroids. Improved DOX accumulation obtained with MXO and PTX resulted in respectively 1.4- and 2.2-fold higher LDH release compared to spheroids treated only with DOX. Interestingly, although preincubation with SCL did not promote DOX, MDOX or LDOX accumulation, LDH release of spheroids cultured with SCL before DOX treatment was 2.5-fold higher than ones treated only with DOX. While LDOX toxicity was not increased by any pretreatment, viability of cells incubated with MDOX decreased further when pretreated with PTX and MXO (1.5- and 1.8-fold compared to MDOX respectively). Low LDOX toxicity is in agreement with [7] and may be due to insufficient DOX release[8]. We report for the first time use of spheroids to test DOX toxicity potentiation as free, micellar or liposomal forms. Out of the six inducers tested, three (PTX, MXO and SCL) enhanced DOX accumulation and/or toxicity. Results suggest different mechanisms of priming for these three drugs and support sequential chemotherapy with PTX, MXO or SCL and DOX. Acknowledgments: This work was supported by grant CCNE IUCA151881 to V.P. Torchilin.