Three-dimensional tumor spheroids for in vitro analysis of bacteria as gene delivery vectors in tumor therapy.

Annika Osswald,Kerstin Otte,Verena Grimm,Zhongke Sun,Astrid M Westendorf,Grace Ampem,Karin Riegel,Christian U Riedel,Peter Dürre,Wolfgang Sommergruber

doi:10.1186/s12934-015-0383-5

Abstract

BackgroundSeveral studies in animal models demonstrated that obligate and facultative anaerobic bacteria of the genera Bifidobacterium, Salmonella, or Clostridium specifically colonize solid tumors. Consequently, these and other bacteria are discussed as live vectors to deliver therapeutic genes to inhibit tumor growth. Therapeutic approaches for cancer treatment using anaerobic bacteria have been investigated in different mouse models. In the present study, solid three-dimensional (3D) multicellular tumor spheroids (MCTS) of the colorectal adenocarcinoma cell line HT-29 were generated and tested for their potential to study prodrug-converting enzyme therapies using bacterial vectors in vitro.ResultsHT-29 MCTS resembled solid tumors displaying all relevant features with an outer zone of proliferating cells and hypoxic and apoptotic regions in the core. Upon incubation with HT-29 MCTS, Bifidobacterium bifidum S17 and Salmonella typhimurium YB1 selectively localized, survived and replicated in hypoxic areas inside MCTS. Furthermore, spores of the obligate anaerobe Clostridium sporogenes germinated in these hypoxic areas. To further evaluate the potential of MCTS to investigate therapeutic approaches using bacteria as gene delivery vectors, recombinant bifidobacteria expressing prodrug-converting enzymes were used. Expression of a secreted cytosine deaminase in combination with 5-fluorocytosine had no effect on growth of MCTS due to an intrinsic resistance of HT-29 cells to 5-fluorouracil, i.e. the converted drug. However, a combination of the prodrug CB1954 and a strain expressing a secreted chromate reductase effectively inhibited MCTS growth.ConclusionsCollectively, the presented results indicate that MCTS are a suitable and reliable model to investigate live bacteria as gene delivery vectors for cancer therapy in vitro.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0383-5) contains supplementary material, which is available to authorized users.

Highlights

Several studies in animal models demonstrated that obligate and facultative anaerobic bacteria of the genera Bifidobacterium, Salmonella, or Clostridium colonize solid tumors
In order to investigate bacterial tumor targeting in vitro, multicellular tumor spheroids (MCTS) of the colorectal cancer cell line HT-29 were established as a 3D model for solid tumors
HT-29 MCTS displayed an intense purple hematoxylin staining in the outer layers of the MCTS, which gradually fainted into a pink eosin staining towards the core (Additional file 1: Figure S1A)

Summary

Introduction

Several studies in animal models demonstrated that obligate and facultative anaerobic bacteria of the genera Bifidobacterium, Salmonella, or Clostridium colonize solid tumors. Several studies in animal models have shown that bifidobacteria selectively colonize and replicate in solid tumors following oral, intravenous, or intratumoral application [16, 17] Due to their non-pathogenic nature and broad use as probiotics, bifidobacteria are promising candidates as live vectors for delivery and expression of therapeutic genes to inhibit tumor growth. Most bifidobacteria are highly resistant to genetic manipulation and only a very limited number of strains have been modified to express genes relevant for tumor therapy [18,19,20] Another group of Gram-positive, strictly anaerobic bacteria widely used for tumor targeting strategies are spore-forming Clostridium sp. Tumors did not recur in approximately 30 % of the animals [25, 28]

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Discussion

Conclusion