Magnetic Fluid Hyperthermia as Treatment Option for Pancreatic Cancer Cells and Pancreatic Cancer Organoids

Abstract

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a cancer with a meager prognosis due to its chemotherapy resistance. A new treatment method may be magnetic fluid hyperthermia (MFH). Magnetoliposomes (ML), consisting of superparamagnetic iron oxide nanoparticles (SPION) stabilized with a phospholipid-bilayer, are exposed to an alternating magnetic field (AMF) to generate heat. To optimize this therapy, we investigated the effects of MFH on human PDAC cell lines and 3D organoid cultures.

Material and methods: ML cytotoxicity was tested on Mia PaCa-2 and PANC-1 cells and on PDAC 3D organoid cultures, generated from resected tissue of patients. The MFH was achieved by AMF application with an amplitude of 40-47 kA/m and a frequency of 270 kHz. The MFH effect on the cell viability of the cell lines and the organoid cultures was investigated at two different time points. Clonogenic assays evaluated the impairment of colony formation. Altering ML set-ups addressed differences arising from intra- vs extracellular ML locations.

Results: Mia PaCa-2 and PANC-1 cells showed no cytotoxic effects at ML concentrations up to 300 µg(Fe)/mL and 225 µg(Fe)/mL, respectively. ML at a concentration of 225 µg(Fe)/mL were also non-toxic for PDAC organoid cultures. MFH treatment using exclusively extracellular ML presented the highest impact on cell viability. Clonogenic assays demonstrated remarkable impairment as long-term outcome in MFH-treated PDAC cell lines. Additionally, we successfully treated PDAC organoids with extracellular ML-derived MFH, resulting in notably reduced cell viabilities 2h and 24 h post treatment. Still, PDAC organoids seem to partly recover from MFH after 24 h as opposed to conventional 2D-cultures.

Conclusion: Treatment with MFH strongly diminished pancreatic cancer cell viability in vitro, making it a promising treatment strategy. As organoids resemble the more advanced in vivo conditions better than conventional 2D cell lines, our organoid model holds great potential for further investigations.

Keywords: PDAC; SPION; magnetic fluid hyperthermia; magnetic nanoparticles; organoids; pancreatic cancer.

Figure 1

Experimental set-up of 2D cell culture MFH trials.

Figure 2

Cytotoxicity testing of increasing ML concentrations ranging from 0 µg(Fe)/mL to 450 µg(Fe)/mL on human pancreatic ductal cancer PANC-1 (A) and Mia PaCa-2 (B) cells revealed no cytotoxic effect at concentrations of up to 300 µg(Fe)/mL and 225 µg(Fe)/mL, respectively (*p < 0.05, **p < 0.01, ***p < 0.001). All samples were normalized to the control group of 0 µg (Fe)/mL. Cell viability of greater than 100% is due to the normalization with respect to the control group.

Figure 3

Cell viability of Mia PaCa-2 and PANC-1 cells at 0 hours (A and B) and 24 hours (C and D) after MFH treatment. Extracellular MFH (´extracellular ML + AMF´) accounted for the most prominent decrease in cell viability at 0 hours (A and B) as well as at 24 hours (C and D) after the treatment. This applied to both cell lines. (**p < 0.01, ***p < 0.001).

Figure 4

Survival fractions of MFH-treated Mia PaCa-2 (A) and PANC-1 (B) as quantified by Clonogenic Assay after treatment. MFH treatment resulted in marked decrease in clonogenic potential of both cell lines for all set-ups with ML. Yet, this effect was most pronounced for samples treated with intra- and extracellular MFH as well as extracellular MFH. (**p < 0.01, ***p < 0.001).

Figure 5

Heating characteristics of two different Geltrex™ set-ups.

Figure 6

(A) Microscopical image of human patient-derived PDAC organoids portraying the characteristic 3D organoid structure. (B) Microscopical image of a human patient-derived PDAC organoid after 24 hours of incubation with nanoparticles.

Figure 7

Cell viability testing of patient-derived PDAC organoids (PANCO-9a) after 24-hour incubation with ML at a concentration of 225 µg(Fe)/mL showed neglectable cytotoxic effects. (***p < 0.001).

Figure 8

Exemplary histological images of ML interacting with PDAC organoids. Prussian blue staining highlights ML in blue in the ´positive control´ sample. Immunohistochemistry staining for Ki67 and CC3 showed no significant alterations in cell proliferation rate and apoptosis rate in PDAC organoids upon ML treatment in comparison to native PDAC organoids. Exemplary organoids for each set-up are depicted.

Figure 9

Cell survival of patient-derived PDAC organoids (PANCO-9a) upon MFH treatment. MFH-treated patient-derived PDAC organoids showed a decrease in cell viability of 48% at 2 hours post treatment (A) and of 13% 24 hours post treatment (B), respectively. All samples were normed to the control: ´no ML´.