Ultrasound-induced Cavitation enhances the efficacy of Chemotherapy in a 3D Model of Pancreatic Ductal Adenocarcinoma with its microenvironment

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is supported by a complex microenvironment whose physical contribution to chemoresistance could be overcome by ultrasound (US) therapy. This study aims to investigate the ability of US-induced inertial cavitation in association with chemotherapy to alter tumor cell viability via microenvironment disruption. For this purpose, we used a 3D-coculture PDAC model partially mimicking the tumor and its microenvironment. Coculture spheroids combining DT66066 cells isolated from KPC-transgenic mice and murine embryonic fibroblasts (iMEF) were obtained by using a magnetic nanoshuttle method. Spheroids were exposed to US with incremental inertial cavitation indexes. Conditions studied included control, gemcitabine, US-cavitation and US-cavitation + gemcitabine. Spheroid viability was assessed by the reduction of resazurin and flow cytometry. The 3D-coculture spheroid model incorporated activated fibroblasts and produced type 1-collagen, thus providing a partial miniature representation of tumors with their microenvironment. Main findings were: (a) Gemcitabine (5 μM) was significantly less cytotoxic in the presence of KPC/iMEFs spheroids compared with KPC (fibroblast-free) spheroids; (b) US-induced inertial cavitation combined with Gemcitabine significantly decreased spheroid viability compared to Gemcitabine alone; (c) both cavitation and chemotherapy affected KPC cell viability but not that of fibroblasts, confirming the protective role of the latter vis-à-vis tumor cells. Gemcitabine toxicity is enhanced when cocultured spheroids of KPC and iMEF are exposed to US-cavitation. Although the model used is only a partial representation of PDAC, this experience supports the hypothesis that US-inertial cavitation can enhance drug penetration and cytotoxicity by disrupting PDAC microenvironment.

Figure 1

Immuno-fluorescence imaging of KPCF spheroid at day 3 of growth. KPC and iMEFs respectively expressed mCherry (red) and Green Fluorescent Protein (GFP) for clear immuno-fluorescent demarcation of both components.

Figure 2

Histological section (5 microns) of a KPCF spheroid after 10 days of incubation. Staining with HE (Hematoxylin Eosin) and Masson’s Trichrome. Collagen fibers appear in blue. Blackish deposits correspond to residual magnetic nanoparticles.

Figure 3

Ratio of viability between KPCF and KPC spheroids treated with Gem compared to control. Viability was measured 24 h after low-dose (5 μM) Gemcitabine incubation: Comparison of KPCF (KPC + fibroblasts) vs KPC (monotypic) spheroid models (n = 6 spheroids for each condition).

Figure 4

KPCF viability after various treatment conditions: Gem/US/US + Gem. (a) KPCF viability after US CI 14 treatment combined or not to Gem exposure. N = 6 spheroids for each condition. (b) KPCF viability after US CI 20 treatment combined or not to Gem exposure. N = 6 spheroids for each condition. (c) KPCF viability after US CI 26 treatment combined or not to Gem exposure. N = 6 spheroids for each condition.

Figure 5

KPCF spheroid viability after US CI 20 treatment and Gem exposure. Cytometer analysis. Analysis based on 4 repeat experiments with 6 spheroids pooled for each condition.

Figure 6

Example of the cytometer analysis assessing the viability measured by BV 510-A of KPC cells from dissociated KPCF spheroids after various treatments.

Figure 7

Coculture protocol/Formation of tumor spheroids/Treatment protocol design.

Figure 8

Spheroid position in the modified Eppendorf® tube.