Flura-Seq Identifies In Situ Transcriptomes of Micrometastases

Understanding the biology of rare cell populations in the context of their microenvironment requires an accurate analysis of the transcriptomes of these cells as expressed in situ. We developed fluorouracil-tagged RNA sequencing (Flura-seq) to characterize the transcriptomes of small cell subpopulations from a whole organ in model systems. The method utilizes cytosine deaminase (CD), which converts the non-natural pyrimidine base fluorocytosine to fluorouracil. Expression of S. cerevisiae CD in cells of interest and exposure to fluorocytosine generates fluorouracil, which is metabolically incorporated into newly synthesized RNAs. The fluorouracil-tagged RNAs can then be immunopurified and sequenced. We applied Flura-seq to define the transcriptome of human breast cancer xenografts, representing as few as 0.003% of host organ cell population during the early stages of metastatic colonization of mouse lungs. The robustness, simplicity and lack of toxicity of Flura-seq make this tool broadly applicable to many studies in developmental, regenerative, and cancer biology. RNAs were tagged withfluorouracil, and the tagged RNAs were immunopurified before sequencing. MDA_Con-Dox = MDA231 control cells that does not express CD/UPRT MDA_FC50uM4hr = MDA231 cells treated with Doxycycline for 24 hours to express CD/UPRT and further treated with 50 uM 5-fluorocytosine for 4 hours. MDA_FC50uM12hr = MDA231 cells treated with Doxycycline for 24 hours to express CD/UPRT and further treated with 50 uM 5-fluorocytosine for 12 hours. MDA_FC250uM4hr = MDA231 cells treated with Doxycycline for 24 hours to express CD/UPRT and further treated with 250 uM 5-fluorocytosine for 4 hours. MDA_FC250uM4hr = MDA231 cells treated with Doxycycline for 24 hours to express CD/UPRT and further treated with 250 uM 5-fluorocytosine for 12 hours. MDA_SBCon = MDA231 cells treated with 2.5 uM SB-505124 for 150minutes. MDA_TGFBCon = MDA231 cells treated with 200 pM TGFB for 150minutes. MDA_SB_IP = MDA231 cells treated with Doxycyline for 24 hours to express CD/UPRT, then with 250 uM of 5-fluorocytosine for 30 minutes before adding 2.5 uM of SB-505124 for additional 150 minutes, and the mRNAs from the cells were immunoprecipitated with anti-BrdU antibody. MDA_TGFB_IP = MDA231 cells treated with Doxycyline for 24 hours to express CD/UPRT, then with 250 uM of 5-fluorocytosine for 30 minutes before adding 200 pM of TGFB for additional 150 minutes, and the mRNAs from the cells were immunoprecipitated with anti-BrdU antibody. MDA_msLung_4h Input = MDA231 cells injected in mouse lung through tail vien, and 4 weeks after the injection CD/UPRT was induced by feeding Doxycycline diet for 2-3 days, and 250 mg/kg 5-fluorocytosine was injected intraperitoneally and 125 mg/kg thymine subcutaneously, and 4 hours later mouse lungs were harvested, homogenized, mRNAs were isolated and sequenced. MDA_msLung_4h IP = MDA231 cells injected in mouse lung through tail vien, and 4 weeks after the injection CD/UPRT was induced by feeding Doxycycline diet for 2-3 days, and 250 mg/kg 5-fluorocytosine was injected intraperitoneally and 125 mg/kg thymine subcutaneously, and 4 hours later mouse lungs were harvested, homogenized, mRNAs were isolated, immunopurified with anti-BrdU antibody and sequenced. MDA_msLung_12h Input = MDA231 cells injected in mouse lung through tail vien, and 4 weeks after the injection CD/UPRT was induced by feeding Doxycycline diet for 2-3 days, and 250 mg/kg 5-fluorocytosine was injected intraperitoneally and 125 mg/kg thymine subcutaneously, and 12 hours later mouse lungs were harvested, homogenized, mRNAs were isolated and sequenced. MDA_msLung_12h IP = MDA231 cells injected in mouse lung through tail vien, and 4 weeks after the injection CD/UPRT was induced by feeding Doxycycline diet for 2-3 days, and 250 mg/kg 5-fluorocytosine was injected intraperitoneally and 125 mg/kg thymine subcutaneously, and 12 hours later mouse lungs were harvested, homogenized, mRNAs were isolated, immunopurified with anti-BrdU antibody and sequenced.

要在微环境（microenvironment）背景下解析稀有细胞群体的生物学特性，需对这些细胞的原位表达转录组（transcriptome）进行精准分析。我们开发了氟尿嘧啶标记RNA测序（fluorouracil-tagged RNA sequencing, Flura-seq）技术，用于在模型系统中解析完整器官内小型细胞亚群的转录组特征。
该技术利用胞嘧啶脱氨酶（cytosine deaminase, CD），可将非天然嘧啶碱基氟胞嘧啶转化为氟尿嘧啶。在目标细胞中表达酿酒酵母（S. cerevisiae）来源的CD，并将细胞暴露于氟胞嘧啶，即可生成氟尿嘧啶，后者可通过代谢途径整合进入新合成的RNA分子中。随后，可通过免疫纯化获取氟尿嘧啶标记的RNA并进行测序。
我们将Flura-seq技术应用于人乳腺癌异种移植瘤（xenografts）的转录组解析，该移植瘤在小鼠肺脏转移定植早期仅占宿主器官细胞群体的0.003%。Flura-seq技术具有稳健性强、操作简便且无毒性的优势，可广泛应用于发育生物学、再生生物学以及癌症生物学领域的诸多研究。
所有RNA均经氟尿嘧啶标记，标记后的RNA在测序前完成免疫纯化。
各样本详情如下：
MDA_Con-Dox：未表达CD/UPRT的MDA231对照细胞
MDA_FC50uM4hr：经24小时多西环素诱导表达CD/UPRT，随后用50 μM 5-氟胞嘧啶处理4小时的MDA231细胞
MDA_FC50uM12hr：经24小时多西环素诱导表达CD/UPRT，随后用50 μM 5-氟胞嘧啶处理12小时的MDA231细胞
MDA_FC250uM4hr：经24小时多西环素诱导表达CD/UPRT，随后用250 μM 5-氟胞嘧啶处理4小时的MDA231细胞
MDA_FC250uM4hr：经24小时多西环素诱导表达CD/UPRT，随后用250 μM 5-氟胞嘧啶处理12小时的MDA231细胞
MDA_SBCon：用2.5 μM SB-505124处理150分钟的MDA231细胞
MDA_TGFBCon：用200 pM 转化生长因子β（transforming growth factor β, TGFB）处理150分钟的MDA231细胞
MDA_SB_IP：经24小时多西环素诱导表达CD/UPRT，先用250 μM 5-氟胞嘧啶处理30分钟，再加入2.5 μM SB-505124继续处理150分钟，随后用抗BrdU抗体免疫沉淀细胞内mRNA的MDA231细胞
MDA_TGFB_IP：经24小时多西环素诱导表达CD/UPRT，先用250 μM 5-氟胞嘧啶处理30分钟，再加入200 pM TGFB继续处理150分钟，随后用抗BrdU抗体免疫沉淀细胞内mRNA的MDA231细胞
MDA_msLung_4h Input：通过尾静脉注射到小鼠肺脏的MDA231细胞；接种4周后，用多西环素饮食喂养2-3天以诱导CD/UPRT表达，随后腹腔注射250 mg/kg 5-氟胞嘧啶，皮下注射125 mg/kg 胸腺嘧啶，4小时后处死小鼠并采集肺脏，将肺脏匀浆后分离mRNA并进行测序的样本
MDA_msLung_4h IP：通过尾静脉注射到小鼠肺脏的MDA231细胞；接种4周后，用多西环素饮食喂养2-3天以诱导CD/UPRT表达，随后腹腔注射250 mg/kg 5-氟胞嘧啶，皮下注射125 mg/kg 胸腺嘧啶，4小时后处死小鼠并采集肺脏，将肺脏匀浆后分离mRNA，经抗BrdU抗体免疫纯化后进行测序的样本
MDA_msLung_12h Input：通过尾静脉注射到小鼠肺脏的MDA231细胞；接种4周后，用多西环素饮食喂养2-3天以诱导CD/UPRT表达，随后腹腔注射250 mg/kg 5-氟胞嘧啶，皮下注射125 mg/kg 胸腺嘧啶，12小时后处死小鼠并采集肺脏，将肺脏匀浆后分离mRNA并进行测序的样本
MDA_msLung_12h IP：通过尾静脉注射到小鼠肺脏的MDA231细胞；接种4周后，用多西环素饮食喂养2-3天以诱导CD/UPRT表达，随后腹腔注射250 mg/kg 5-氟胞嘧啶，皮下注射125 mg/kg 胸腺嘧啶，12小时后处死小鼠并采集肺脏，将肺脏匀浆后分离mRNA，经抗BrdU抗体免疫纯化后进行测序的样本