Functional Assay for Measuring Bacterial Degradation of Gemcitabine Chemotherapy.
Functional Assay for Measuring Bacterial Degradation of Gemcitabine Chemotherapy.
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DOI：
10.21769/bioprotoc.4797
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发表时间：
2023-09-05
期刊：
影响因子：
0.8
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中科院分区：
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宿主微生物组的药物生物转化可以影响治疗的治疗成功。在癌症的背景下，药物降解可以通过肿瘤内细菌在靶向肿瘤的微环境内发生。在胰腺癌中，对一线化疗吉西他滨的耐药性增加被认为是由肿瘤微生物组的药物降解引起的。这种细菌-药物相互作用突出了开发用于监测细菌吉西他滨分解的快速测定的需要。虽然化学方法，如高效液相色谱法适用于这一任务，但它们需要专门的设备和专业知识，并且产量有限。基于功能性细胞的测定代表了用于执行该任务的替代方法。我们开发了一种功能测定法，使用高度敏感的细菌报告菌株来监测细菌吉西他滨分解的速率。我们的方法依赖于标准实验室设备，可以高通量实施，以同时监测数百种菌株的药物分解。该功能测定可容易地适用于监测其他药物的降解。 通过孵育降解药物的细菌并随后检测报告菌株在过滤上清液上的生长来定量吉西他滨降解。使用经遗传工程改造为非降解菌株且对吉西他滨高度敏感的优化报告菌株。一种在微孔板中进行的高通量测定法，可进行调整以鉴定具有快速或慢速吉西他滨降解速率的细菌。可以将测定结果与具有已知药物浓度的标准曲线的结果进行比较，以量化降解速率。
Drug biotransformation by the host microbiome can impact the therapeutic success of treatment. In the context of cancer, drug degradation can take place within the microenvironment of the targeted tumor by intratumor bacteria. In pancreatic cancer, increased chemo-resistance against the frontline chemotherapy gemcitabine is thought to arise from drug degradation by the tumor microbiome. This bacterial–drug interaction highlights the need for developing rapid assays for monitoring bacterial gemcitabine breakdown. While chemical approaches such as high-performance liquid chromatography are suitable for this task, they require specialized equipment and expertise and are limited in throughput. Functional cell-based assays represent an alternate approach for performing this task. We developed a functional assay to monitor the rate of bacterial gemcitabine breakdown using a highly sensitive bacterial reporter strain. Our method relies on standard laboratory equipment and can be implemented at high throughput to monitor drug breakdown by hundreds of strains simultaneously. This functional assay can be readily adapted to monitor degradation of other drugs. Key features Quantification of gemcitabine breakdown by incubating bacteria that degrades the drug and subsequently testing the growth of a reporter strain on filtered supernatant. Use of an optimized reporter strain that was genetically engineered to be a non-degrader strain and highly sensitive to gemcitabine. A high-throughput assay performed in microplates that can be adjusted for identifying bacteria with a fast or slow gemcitabine degradation rate. The assay results can be compared to results from a standard curve with known drug concentrations to quantify degradation rate.