Anti-Cancer Effects of Artesunate in Human 3D Tumor Models of Different Complexity

Abstract

The anti-malaria drug Artesunate (ART) shows strong anti-cancer effects in vitro; however, it shows only marginal treatment results in clinical cancer studies. In this study, ART was tested in preclinical 3D cancer models of increasing complexity using clinically relevant peak plasma concentrations to obtain further information for translation into clinical use. ART reduced cell viability in HCT-116 and HT-29 derived cancer spheroids (p < 0.001). HCT-116 spheroids responded dose-dependently, while HT-29 spheroids were affected more strongly by ART than by cytostatics (p < 0.001). HCT-116 spheroids were chemo-sensitized by ART (p < 0.001). In patient-derived cancer spheroids (PDCS), ART led to inhibition of cell viability in 84.62% of the 39 samples tested, with a mean inhibitory effect of 13.87%. Viability reduction of ART was 2-fold weaker than cytostatic monotherapies (p = 0.028). Meanwhile, tumor-stimulation of up to 16.30% was observed in six (15.38%) PDCS-models. In 15 PDCS samples, ART modulated chemotherapies in combined testing, eight of which showed chemo-stimulation (maximum of 36.90%) and seven chemo-inhibition (up to 16.95%). These results demonstrate that ART's anti-cancer efficacy depends on the complexity of the tumor model used. This emphasizes that cancer treatment with ART should be evaluated before treatment of the individual patient to ensure its benefits and prevent unwanted effects.

Keywords: 3D cancer model; Artesunate; artemisinin-derivatives; cancer spheroid model; neoplasia.

Figure 1

Cell line derived cancer spheroids treated with 15 µg/mL of Artesunate (B,D) or the cor-responding solvent control (A,C) for 72 h. (A,B) HCT-116 derived cancer spheroids. (C,D) HT-29 derived cancer spheroids. Photographs in the corner show spheroids at 40× magnification, while larger photographs show spheroids at 100× magnification; white bar in the corner shows a length of 200 µm.

Figure 2

Anti-cancer effects of Artesunate (ART) in different peak plasma concentrations compared to guideline recommended chemotherapies in HCT-116 (A) and HT-29 (B) derived cancer spheroids. 5FU, 5-Fluorouracil; FO, 5FU+Oxaliplatin; FI, 5FU+Irinotecan; SC, solvent control. * p < 0.01.

Figure 3

Chemo-sensitization induced by Artesunate (ART) at a PPC of 0.74 µg/mL (A) and 15 µg/mL (B) in HCT-116 derived cancer spheroids. 5FU, 5-Fluorouracil; FO, 5FU + Oxaliplatin; FI, 5FU + Irinotecan; SC, solvent control. * p < 0.001.

Figure 4

Combination therapy of 5FU, FO and FI with Artesunate (ART) at 0.74 µg/mL (A) and 15 µg/mL (B) in HT-29 derived cancer spheroids. 5FU, 5-Fluorouracil; FO, 5FU + Oxaliplain; FI, 5FU + Irinotecan; SC, Solvent Control. * p < 0.01.

Figure 5

Patient-derived cancer spheroids treated with Solvent Control of Artesunate (A,C) or 0.74 µg/mL of Artesunate (B,D); Photographs (A,B) show spheroids at 40× magnification, photographs (C,D) show them at 100× magnification; white bar in the corner shows a length of 100 µm.

Figure 6

Effect of Artesunate (ART) on patient-derived cancer spheroids (PDCS) and peripheral blood mononuclear cells (PBMC). For reference, chemotherapies (CTx) are shown as single agents (CTx Mono), doublets (CTx Double), and triplets (CTx Triple). * p < 0.05; SC, Solvent Control; n, number of tests performed. Bars represent the mean.

Figure 7

Artesunate (ART) treatment of PBMC induces hemolysis in a dose-dependent manner. (A) solvent control corresponding to the ART PPC 15 µg/mL. (B) ART PPC 0.74 µg/mL. (C) ART PPC 3.26 µg/mL. (D) ART PPC 15 µg/mL. Photographs show PBMCs at 100× magnification; white bar in the corner shows a length of 100 µm.

Figure 8

Chemo-stimulating (A) and Chemo-reducing effects (B) of Artesunate in patient-derived cancer spheroids. ART, Artesunate; CTx, Chemotherapy; SC, Solvent Control.

Figure 9

Viability-modulating effects of Artesunate in patient derived cancer spheroids. Pat 1 (A), strongest inhibition of cell viability, recurrent cervical cancer. Pat 2 (B), strongest stimulation of cell viability, recurrent liposarcoma; Pat 3 (C), inhibition of cell viability equally strong as tested cytostatics, primary metastatic breast cancer. ART, Artesunate; Cis, Cisplatin; MTX, Methotrexate; Doxo, Doxorubicin; Trab, Trabectedin; Pem, Pembrolizumab; Gem, Gemcitabine; SC, Solvent Control; * p < 0.001.

Figure 10

Chemo-modulating effects of Artesunate in patient-derived cancer spheroids. Pat 4 (A), chemo-sensitization, recurrent glioblastoma. Pat 5 (B), chemo-sensitization, metastatic gastric cancer. Pat 6 (C), chemo-reduction, primary ovarian cancer. ART, Artesunate; Tem, Temozolomide; FI, 5-Fluorouracil + Irinotecan; CP, Carboplatin + Paclitaxel; SC, Solvent Control; * p < 0.001.