Cancer cell spheroids as a model to evaluate chemotherapy protocols

Abstract

To determine whether the spheroid culture can be used to evaluate drug efficacy, we have evaluated the toxicity of free or carrier-associated doxorubicin as a single drug or in combination with other antineoplastic agents using the spheroid cultures of drug-resistant cancer cells. Paclitaxel, cisplatin, dexamethasone, mitoxantrone, sclareol or methotrexate were used in combination with doxorubicin. The effect of the treatment protocols on free, micellar and liposomal doxorubicin accumulation in spheroids and on resulting toxicity was evaluated by fluorescence and lactate dehydrogenase release, respectively. Enhanced doxorubicin accumulation and toxicity were observed after spheroid pretreatment with mitoxantrone or paclitaxel. Effects of the drug combination with doxorubicin were sequence dependent, use of doxorubicin as the first drug being the least inducer of toxicity. Finally, spheroids were recognized by a cancer cell-specific antibody. Our results suggest the usefulness of spheroids to evaluate chemotherapy combinations.

Figure 1. The effect of cytotoxic doses of priming drugs on NCI-ADR-RES spheroids. NCI-ADR-RES spheroids were cultured with sclareol (SCL), dexamethasone (DXM), cisplatin (CIS), methotrexate (MTX), paclitaxel (PTX) or mitoxantrone (MXO) for 48 h before determination of LDH release with a Cytotox 96 cell viability kit (A) and apoptosis induction by a Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (B). Concentrations used are indicated in micromolars. LDH release was normalized to untreated cells cultured in the same conditions. Pretreated spheroids were fixed and submitted to TUNEL assay: A, untreated spheroids; B, DNase I (positive control); C, SCL; D, MTX; E, CIS; F, DXM; G, MXO; H, PTX. Scale bar represents 200 µm. Data represent the mean ± SD, n = 3. Student’s t-test; *p < 0.05, **p < 0.01 compared with untreated cells.

Figure 2. Enhancement of doxorubicin accumulation and toxicity after pretreatment of NCI-AR-RES spheroids. NCI-ADR-RES spheroids were preincubated with cytotoxic doses of priming drugs before the addition of doxorubicin (DOX, 100 µM): sclareol (SCL + DOX), dexamethasone (DXM + DOX), cisplatin (CIS + DOX), methotrexate (MTX + DOX), paclitaxel (PTX + DOX), mitoxantrone (MXO + DOX) or doxorubicin alone (DOX). The accumulation of doxorubicin (black bars) was quantitated by fluorescence and was expressed in nanomoles of doxorubicin per mg of protein. LDH release (white bars) was measured (Fig. 1). Same concentrations as in Figure 1 were used. Data represent the mean ± SD, n = 3. Student’s t-test; *p < 0.05, **p < 0.01 compared with DOX alone group.

Figure 3. Enhancement of micellar doxorubicin accumulation and toxicity after pretreatment of NCI-AR-RES spheroids. NCI-ADR-RES spheroids were preincubated with cytotoxic doses of priming drugs before addition of micellar doxorubicin (100 µM): sclareol (SCL + MDOX), dexamethasone (DXM + MDOX), cisplatin (CIS + MDOX), methotrexate (MTX + MDOX), paclitaxel (PTX + MDOX), mitoxantrone (MXO + MDOX) or media as control (MDOX). The accumulation of doxorubicin (black bars) was quantitated (see Fig. 2). LDH release (white bars) was measured (see Fig. 1). Data represent the mean ± SD, n = 3. Student’s t-test; *p < 0.05, **p < 0.01 compared with MDOX alone group.

Figure 4. Cytotoxicity of empty and doxorubicin-loaded micelles. NCI-ADR-RES spheroids were preincubated with cytotoxic doses of priming drugs before addition of PEG 2000 micelles (MIC) either empty (white bars) or DOX-loaded (black bars). LDH release was measured with the Cytotox 96 cell viability kit and normalized to untreated cells. Data represent the mean ± SD, n = 3. Student’s t test; *p < 0.05, **p < 0.01 between empty and DOX-loaded micelles.

Figure 5. Enhancement of liposomal doxorubicin accumulation and toxicity after pretreatment of NCI-ADR-RES spheroids. NCI-ADR-RES spheroids were preincubated with cytotoxic doses of priming drugs before addition of Lipo-Dox (LDOX, 100 µM): sclareol (SCL + LDOX), dexamethasone (DXM + LDOX), cisplatin (CIS + LDOX), methotrexate (MTX + LDOX), paclitaxel (PTX + LDOX), mitoxantrone (MXO + LDOX) or LDOX alone (LDOX). The accumulation of doxorubicin (black bars) was quantitated (Fig. 2). LDH release (white bars) was measured (Fig. 1). Data represent the mean ± SD, n = 3. Student’s t-test; **p < 0.01 compared with unprimed group.

Figure 6. Schedule-dependency of drug combinations on NCI-ADR-RES spheroids. Three regimens of drug combinations were evaluated, priming (inducer drug for 48 h before DOX 24 h, white bars), reverse priming (DOX for 48 h as a first line agent before second drug for 24 h, gray bars) and co-administration of both drugs (black bars). LDH release was measured 72 h after addition of the second drug or their co-administration and was normalized to untreated cells. Data represent the mean ± SD, n = 3. Student’s t-test; *p < 0.05, **p < 0.01 compared with DOX alone.

Figure 7. Binding of 2C5 antibodies. NCI-ADR-RES cells grown as spheroids (A) or monolayers (B) were incubated with 2C5 or control isotype-matching antibody. Solid: untreated cells, gray line: isotype-matching control, black line: 2C5 antibody.