Bone invigorates metstatic seeding [evolving barcode]

Following the removal of implanted mammary tumors, nude mice develop multiple-organ metastases at late stage. The metastases may originate from the primary tumors before the resection surgery, or alternatively, from some established metastases. By multiple approaches, we have proved that bone environment could invigorate cancer cells for further dissemination. this study aims to examine if metastatic dissemination from bone to other sites occurs in natural setting of metastatic spread. We herein apply the rapidly evolving barcode system using homing guide RNA/Cas9 to trace the metastases formation in mouse. hgRNA/Cas9 is a self-targeting Crispr system which allows the mutation occurs in the DNA sequence of guide RNA. Tumor cells wer labelled with doxycycline inducible evolving barcoding system. Upon doxycycline treatment the DNA sequence of hgRNA accumulate mutations with time. The diversity of barcodes in each lesion can infer the timeing of seeding while the mutation patterns of barcodes suggest the phylogenetic correlation of metastases. Several findings were made on this study. First, at the terminal stage, multi-organ metastases are not genetically grouped according to sites of metastases. Nonnegative Matrix Factorization (NMF) analysis of mutant barcodes suggested the early disseminated metastases, which have highest level of Shannon entropy, were featured with a common cluster of mutant barcodes irrespective of their locations. Second, most metastases are potentially multiclonal as indicated by multiple clusters of independent mutant barcodes. Third, when we use Shannon entropy as an index of metastasis age , putative parent-child relationship between metastases with unique mutant barcodes clearly exemplified secondary metastatic seeding from bone to other organs. Finally, we did not observe a clear correlation between tumor burden and Shannon entropy across different metastases, suggesting that putative parental metastases might remain small after seeding further metastases. Overall design: Nude mice or C57BL/6 mice were implanted with barcoded MDA-MB-231 or AT-3 cells, respectively. the mammary tumors were removed when the tumor size reached 1.2cm, and a dose of doxycycline were administated to the mice weekly via intra-peritoneal injection for 5 weeks in nude mice or 4 weeks in C57-BL/6 mice. the metastatic lesions were then dissected with assistance of ex vivo BLI imaging and the DNA of each metastases were isolated. The barcodes from DNA samples were then targeted sequenced using Illumina NextSeq 500/550. Mutation events were categorized from the sequence of barcodes and subjected to the following bioinformatic analysis.

在切除植入的乳腺肿瘤后，裸鼠（nude mice）会在疾病晚期出现多器官转移。此类转移灶既可源自切除术前的原发肿瘤，也可来自已形成的转移定植灶。本研究团队通过多种实验手段证实，骨微环境可激活癌细胞，促进其进一步扩散转移。本研究旨在探究在自然转移进程中，是否存在从骨转移灶向其他器官的扩散传播现象。本研究采用基于归巢向导RNA（homing guide RNA, hgRNA）/Cas9的快速进化条形码系统，以追踪小鼠体内的转移灶形成过程。hgRNA/Cas9是一种自靶向CRISPR（Clustered Regularly Interspaced Short Palindromic Repeats）系统，可在向导RNA的DNA序列中引入突变。将肿瘤细胞标记于多西环素（doxycycline）诱导的进化条形码系统中：经多西环素处理后，hgRNA的DNA序列会随时间推移累积突变。每个病灶内条形码的多样性可用于推断肿瘤细胞定植的时间，而条形码的突变模式则可反映不同转移灶之间的系统发育相关性。本研究得到以下几项核心发现：其一，在疾病终末期，多器官转移灶并未按照转移部位形成遗传聚类。对突变条形码的非负矩阵分解（Nonnegative Matrix Factorization, NMF）分析显示，香农熵（Shannon entropy）水平最高的早期播散转移灶，无论其所在解剖部位如何，均以一组共有的突变条形码聚类为特征。其二，从独立突变条形码的多个聚类结果来看，大多数转移灶具有多克隆起源的特性。其三，若以香农熵作为转移灶“年龄”的评估指标，携带独特突变条形码的转移灶之间呈现出明确的亲子关系，这直接证明了从骨转移灶向其他器官的继发性定植转移现象。最后，我们未在不同转移灶中观察到肿瘤负荷与香农熵之间存在明确相关性，这提示亲本转移灶在播散形成次级转移灶后，可能仍维持较小的体积。实验整体设计：分别将携带条形码的MDA-MB-231细胞与AT-3细胞植入裸鼠与C57BL/6小鼠体内。当肿瘤体积达到1.2cm时，切除乳腺原发肿瘤；随后裸鼠每周通过腹腔注射给予多西环素，持续5周，C57BL/6小鼠则给药4周。随后借助离体生物发光成像（ex vivo BLI imaging）技术分离转移灶，并提取每个转移灶的DNA。利用Illumina NextSeq 500/550测序平台对DNA样本中的条形码区域进行靶向测序。从条形码序列中识别突变事件，并开展后续的生物信息学分析。