Mitoxantrone and Mitoxantrone-Loaded Iron Oxide Nanoparticles Induce Cell Death in Human Pancreatic Ductal Adenocarcinoma Cell Spheroids

Abstract

Pancreatic ductal adenocarcinoma is a hard-to-treat, deadly malignancy. Traditional treatments, such as surgery, radiation and chemotherapy, unfortunately are still not able to significantly improve long-term survival. Three-dimensional (3D) cell cultures might be a platform to study new drug types in a highly reproducible, resource-saving model within a relevant pathophysiological cellular microenvironment. We used a 3D culture of human pancreatic ductal adenocarcinoma cell lines to investigate a potential new treatment approach using superparamagnetic iron oxide nanoparticles (SPIONs) as a drug delivery system for mitoxantrone (MTO), a chemotherapeutic agent. We established a PaCa DD183 cell line and generated PANC-1^{SMAD4 (-/-)} cells by using the CRISPR-Cas9 system, differing in a prognostically relevant mutation in the TGF-β pathway. Afterwards, we formed spheroids using PaCa DD183, PANC-1 and PANC-1^{SMAD4 (-/-)} cells, and analyzed the uptake and cytotoxic effect of free MTO and MTO-loaded SPIONs by microscopy and flow cytometry. MTO and SPION-MTO-induced cell death in all tumor spheroids in a dose-dependent manner. Interestingly, spheroids with a SMAD4 mutation showed an increased uptake of MTO and SPION-MTO, while at the same time being more resistant to the cytotoxic effects of the chemotherapeutic agents. MTO-loaded SPIONs, with their ability for magnetic drug targeting, could be a future approach for treating pancreatic ductal adenocarcinomas.

Keywords: 3D cell culture; magnetic drug targeting; nanomedicine; pancreatic ductal adenocarcinoma; superparamagnetic iron oxide nanoparticles.

Figure A1

Confirmation of PDAC^{SMAD4 (−/−)} knock-out mutants by Western blot. Lower panel: GAPDH control shows homogeneous loading of the protein in each line. Upper panel: wt control and non-matching control (lines 1 and 2) show a distinct 61 kDa band of SMAD4. The selected clones (SC) of construct SG1 (lines 3 and 4) do not show any signal, indicating a homogeneous knock-out of the SMAD4 gene in these two clones, while lines 5 and 6 showed much weaker bands slightly lower than the expected size, indicating a truncated variant of the SMAD4 gene and protein.

Figure 1

Generation of PDAC spheroids. Transmission microscopy images of spheroids (a) and cryosections of spheroids after hematoxylin/eosin staining (b) formed by PaCa DD183, PANC-1 and PANC-1^{SMAD4 (−/−)} cells after three, five and seven days of incubation. (c) Growth progression of spheroids between day 3 and 7. Data are expressed as the mean with standard deviation (n = 4–5, with nine replicates each). Statistical significances between spheroid sizes at day 3 and day 7 are indicated with ***. The respective confidential intervals are p ≤ 0.00005 and were calculated via the Student’s t-test. Abbreviations: (1), PaCa DD183; (2), PANC-1; (3), PANC-1^{SMAD4 (−/−)}.

Figure 2

Effect of free MTO on pancreatic spheroids. Transmission microscopy images and quantification of representative spheroids formed by (a,b) PaCa DD183, (c,d) PANC-1 and (e,f) PANC-1^{SMAD4 (−/−)} spheroid cells after three, five and seven days of incubation. On day three, cell culture medium was supplemented with MTO (0, 0.5, 5, 10, 20 and 47.8 µg/mL, respectively). Growth progression was normalized to the spheroid size at day 3. Data are expressed as the mean with standard deviation (n = 4, with three replicates each). Statistical significances between MTO-free and MTO-treated spheroids are indicated with *, ** and ***. The respective confidential intervals are * p ≤ 0.05, ** p ≤ 0.0005 and *** p ≤ 3 × 10⁻⁹, respectively, and were calculated via the Student’s t-test. Abbreviations: MTO, mitoxantrone.

Figure 3

Influence of free MTO on PDAC tumor cell spheroids measured by flow cytometry. (a) Gating strategy. Shown are the representative plots and histograms of cells from PaCa DD183 spheroids after 4 days of treatment with 20 µg/mL MTO (second row) or with the corresponding amount of H₂O (first row). Left panels exemplarily show cell morphology (forward scatter, Fsc, versus side scatter, Ssc), middle panels depict AnnexinV (AxV)-Fitc and propidium iodie (PI) staining, and right histograms represent mitoxantrone (MTO) uptake. (b) Cell viability of PaCa DD183, PANC-1, and PANC-1^{SMAD4 (−/−)} spheroids was determined by Annexin V-FITC/propidium iodide (AxV/PI) staining and analyzed by flow cytometry. Ax−/PI, Ax+/PI and PI+ cells were considered viable, apoptotic and necrotic, respectively. MTO concentrations were used as indicated. Statistical significances are indicated with * and **. The respective confidential intervals are * p ≤ 0.05 and ** p ≤ 0.0005, respectively, and were calculated via the Student’s t-test. Abbreviations: a.u., arbitrary unit; Ssc, side scatter, Fsc, forward scatter; count, detected events; MTO, mitoxantrone.

Figure 4

Effects of nanoparticle-bound MTO on different PDAC tumor cell spheroids. (a–c) Flow cytometric analysis of the viability of cells dissociated from (a) PaCa DD183, (b) PANC-1 and (c) PANC-1^{SMAD4 (−/−)} spheroids after treatment of H₂O, SPIONs, free MTO or SPION-bound MTO was determined by Annexin V-FITC/propidium iodide (AxV/PI) staining. Necrotic, apoptotic and viable cells were determined by PI+, Ax+/PI and Ax−/PI cells, respectively. (d,e) MTO intensity of cells originating from (d) PaCa DD183, (e) PANC-1 and (f) PANC-1^{SMAD4 (−/−)} spheroids, measured by flow cytometry. Statistical significances between control, SPION, MTO and SPION^MTO-treated spheroids are indicated with * and **. The confidential intervals are * p ≤ 0.05 and ** p ≤ 0.0005, respectively, and were calculated via the Student’s t-test. Abbreviations: SPIONs, superparamagnetic iron oxide nanoparticles; MTO, mitoxantrone; a.u., arbitrary unit; ns, not significan4.