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A rapid screening platform to coculture bacteria within tumor spheroids

Nature Protocols volume 17, pages 2216–2239 (2022)Cite this article

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Abstract

The prevalence of tumor-colonizing bacteria along with advances in synthetic biology are leading to a new generation of living microbial cancer therapies. Because many bacterial systems can be engineered to recombinantly produce therapeutics within tumors, simple and high-throughput experimental platforms are needed to screen the large collections of bacteria candidates and characterize their interactions with cancer cells. Here, we describe a protocol to selectively grow bacteria within the core of tumor spheroids, allowing for their continuous and parallel profiling in physiologically relevant conditions. Specifically, tumor spheroids are incubated with bacteria in a 96-well low-adhesion plate followed by a series of washing steps and an antibiotic selection protocol to confine bacterial growth within the hypoxic and necrotic core of tumor spheroids. This bacteria spheroid coculture (BSCC) system is stable for over 2 weeks, does not require specialized equipment and is compatible with time-lapse microscopy, commercial staining assays and histology that uniquely enable analysis of growth kinetics, viability and spatial distribution of both cellular populations, respectively. We show that the procedure is applicable to multiple tumor cell types and bacterial species by varying protocol parameters and is validated by using animal models. The BSCC platform will allow the study of bacteria–tumor interactions in a continuous manner and facilitate the rapid development of engineered microbial therapies.

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Fig. 1: BSCC system to evaluate microbial cancer interactions and therapies.
Fig. 2: Schematic workflow to establish BSCC.
Fig. 3: Overview of the downstream assays and their utilities.
Fig. 4: Coculture with various bacteria and tumor cell types.
Fig. 5: Characterization of bacteria and tumor spheroids in coculture.
Fig. 6: Gene circuit performance in tumor spheroids.
Fig. 7: Therapeutic screening and validation.

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Data availability

The main data discussed in this protocol were generated as part of the studies published in the supporting primary research papers20,21. Source data are provided for Figs. 4–7. Source data are provided with this paper.

Code availability

The computational code used for image analysis is provided in Supplementary Information.

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Acknowledgements

We thank the Herbert Irving Comprehensive Cancer Center Molecular Pathology Shared Resources Facility for help with histological sample processing. We thank W. Mather for assistance with the image analysis. We thank members of the Danino laboratory for reviewing the manuscript. This work was supported by the DoD LC160314 (T.D.), DoD BC160541 (T.D.), NIH 1R01EB029750 (T.D.), NIH F99CA253756 (T.H.) and Honjo International Foundation Scholarship (T.H.).

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Authors and Affiliations

  1. Department of Biomedical Engineering, Columbia University, New York, NY, USA

    Tetsuhiro Harimoto, Dhruba Deb & Tal Danino

  2. Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA

    Tal Danino

  3. Data Science Institute, Columbia University, New York, NY, USA

    Tal Danino

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  1. Tetsuhiro Harimoto
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  2. Dhruba Deb
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T.H. and T.D. conceived and designed the study. T.H., D.D. and T.D. wrote the manuscript.

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Correspondence to Tal Danino.

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Key references using this protocol

Harimoto, T. et al. Proc. Natl. Acad. Sci. USA 116, 9002–9007 (2019): https://doi.org/10.1073/pnas.1820824116

Chien, T. et al. Nat. Biomed. Eng. 6, 94–104 (2022): https://doi.org/10.1038/s41551-021-00772-3

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Supplementary Figs. 1 and 2 and computational code for image analysis

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Harimoto, T., Deb, D. & Danino, T. A rapid screening platform to coculture bacteria within tumor spheroids. Nat Protoc 17, 2216–2239 (2022). https://doi.org/10.1038/s41596-022-00723-5

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