Department of Biomedical Engineering, Columbia University, New York, NY, USA.
Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA.
Nat Protoc. 2022 Oct;17(10):2216-2239. doi: 10.1038/s41596-022-00723-5. Epub 2022 Jul 29.
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.
肿瘤定植细菌的流行以及合成生物学的进步正在推动新一代活体微生物癌症疗法的发展。由于许多细菌系统可以被工程化以在肿瘤内重组产生治疗剂，因此需要简单且高通量的实验平台来筛选大量的候选细菌并表征它们与癌细胞的相互作用。在这里，我们描述了一种在肿瘤球体核心选择性培养细菌的方案，允许在生理相关条件下对其进行连续和并行分析。具体来说，将肿瘤球体与细菌在 96 孔低粘附板中孵育，然后进行一系列洗涤步骤和抗生素选择方案，以将细菌生长限制在肿瘤球体的缺氧和坏死核心内。这种细菌球体共培养（BSCC）系统稳定超过 2 周，不需要专门的设备，并且与延时显微镜、商业染色测定和组织学兼容，分别能够独特地分析细胞群体的生长动力学、活力和空间分布。我们通过改变方案参数证明了该程序适用于多种肿瘤细胞类型和细菌物种，并通过使用动物模型进行了验证。BSCC 平台将允许以连续的方式研究细菌-肿瘤相互作用，并促进工程微生物治疗的快速发展。
